【作者】 郭文静；

【导师】 王金梅； 刘丽军；

【作者基本信息】 河南大学 ， 中药学硕士（专业学位）， 2025， 硕士

【摘要】 山茱萸是山茱萸科植物山茱萸(Cornus officinalis Sieb.et Zucc.)的干燥成熟果肉,有补益肝肾、收敛固涩的功效。现代药理学研究显示,其醇提物和总环烯醚萜苷均有良好的降血糖活性。山茱萸新苷(Cornuside)是从山茱萸的干燥成熟果肉中分离的环烯醚萜苷,具有良好的抗炎、抗氧化、抗细胞凋亡等生理功能。但目前关于其治疗糖尿病的具体作用和机制仍不清楚,因此,本论文旨在体内、外研究山茱萸新苷的降血糖活性及机制,以期为其开发利用提供依据。首先,本研究通过体外实验评估山茱萸新苷对胰岛素抵抗型HepG2细胞(IR-HepG2)葡萄糖代谢调控的影响,重点考察其对细胞葡萄糖摄取能力及糖代谢关键基因表达的影响。采用葡萄糖胺(2-Amino-2-deoxy-D-glucose,Glc N)诱导法构建IR-HepG2细胞模型并采用葡萄糖氧化酶-过氧化物酶(Glucose oxidase-peroxidase,GOD-POD)法检测细胞上清液中葡萄糖含量。结果显示,经山茱萸新苷处理后,IR-HepG2细胞上清液葡萄糖含量显著降低。通过PCR实验检测糖代谢相关基因的表达,结果显示山茱萸新苷能显著逆转Glc N引起的G6PC1、PCK1、GSK3b基因表达上调和SLC2A4、GCK、PFKL基因表达下调。在以上研究的基础上,我们采用高脂饮食(High fat diet,HFD)联合链脲佐菌素(Streptozotocin,STZ)的方法建立C57BL/6小鼠2型糖尿病(Type 2 diabetes mellitus,T2DM)模型。造模成功后给予山茱萸新苷(200、100和50 mg/kg)6周。结果发现山茱萸新苷可以降低T2DM小鼠的空腹血糖、血清中糖化血清蛋白含量;改善T2DM小鼠的体重降低、胰岛素抵抗、葡萄糖耐量和胰岛素耐量;改善T2DM小鼠肝脏组织的病理形态变化,降低肝脏组织中脂质积累。采用16S rDNA和非靶向代谢组学技术进一步探究山茱萸新苷治疗糖尿病的作用机制。结果发现,山茱萸新苷可以升高有益菌群Akkermansia、Bacteroides和Parabacteroides等的相对丰度,降低肠道有害菌Desulfovibrio、Acetatifactor和Vescimonas等的相对丰度,恢复肠道稳态。非靶向代谢组学分析发现,山茱萸新苷可能通过色氨酸代谢(Tryptophan metabolism)、牛磺酸和次牛磺酸代谢(Taurine and hypotaurine metabolism)和初级胆汁酸生物合成(Primary bile acid biosynthesis)等代谢途径,增加黄尿酸(Xanthurenic acid)、十五烷酸(Pentadecanoic acid)和阿魏酸(Ferulic acid)等代谢物水平,降低牛磺熊去氧胆酸(Tauroursodeoxycholic acid)、酮戊二酸(Oxoglutaric acid)和咪唑丙酸(Imidazolepropionic acid)等代谢物水平来发挥作用。通过肠道菌群与代谢组学关联分析发现山茱萸新苷可能主要通过提高Akkermansia的丰度,增强Tryptophan metabolism代谢途径并上调Xanthurenic acid的水平来缓解糖尿病小鼠的代谢损伤。综上所述,山茱萸新苷可以改善IR-HepG2细胞的葡萄糖代谢;改善T2DM小鼠的葡萄糖耐量、胰岛素抵抗、肝脏组织损伤和脂质积累,降低其空腹血糖。山茱萸新苷还可以调节T2DM小鼠盲肠内容物菌群比例和代谢物的水平,提高Akkermansia的丰度,增强Tryptophan metabolism代谢途径并上调Xanthurenic acid的水平来缓解糖尿病小鼠的代谢损伤。更多还原

【Abstract】 Corni Fructus is the dry and ripe fruit of Cornus officinalis Sieb.et Zucc.,which has the effect of toning liver and kidney and astringency.Modern pharmacological studies have shown that both its alcoholic extract and total iridoid glycosides have good hypoglycemic activity.Cornuside is an iridoid glycoside isolated from the dry and mature pulp of Cornus officinalis.Cornuside has good physiological functions such as anti-inflammatory,anti-oxidation,and anti-apoptosis.However,the specific effect and mechanism of cornuside in the treatment of diabetes are still unclear.Therefore,the aim of this study is to investigate the hypoglycemic activity and mechanism of cornuside in vitro and in vivo,and to provide evidence for the development and utilization of cornuside.Firstly,in this study,the effects of cornuside on glucose metabolism in insulin-resistant HepG2 cells(IR-HepG2)were evaluated in vitro,focusing on the glucose uptake ability and the expression of key genes involved in glucose metabolism.The IR-HepG2 cell model was constructed by 2-Amino-2-deoxy-D-glucose(Glc N)induction method,and the glucose content in the cell supernatant was detected by glucose oxidase-peroxidase(GOD-POD)method.The results showed that the glucose content in the supernatant of IR-HepG2 cells was significantly decreased after treatment with cornuside.The expression of genes related to glucose metabolism was detected by PCR,which showed that cornuside could significantly reverse the up-regulation of G6PC1,PCK1 and GSK3b genes and the down-regulation of SLC2A4,GCK and PFKL genes induced by Glc N.On the basis of these studies,we established a C57BL/6 mouse model of Type 2 diabetes mellitus(T2DM)by using High fat diet(HFD)combined with Streptozotocin(STZ).After successful modeling,the rats were treated with cornuside(200,100 and 50 mg/kg)for 6 weeks.The results showed that cornuside could reduce fasting blood glucose,serum glycated serum protein content in T2DM mice.Improved weight loss,insulin resistance,glucose tolerance and insulin tolerance in T2DM mice.It can improve the pathological changes of liver tissue in T2DM mice and reduce the accumulation of lipid in liver tissue.16S rDNA and untargeted metabolomics technology were used to further explore the mechanism of cornuside in the treatment of diabetes.The results showed that cornuside could increase the relative abundance of beneficial bacteria such as Akkermansia,Bacteroides and Parabacteroides.Reducing the relative abundance of intestinal harmful bacteria such as Desulfovibrio,Acetatifactor and Vescimonas and restoring intestinal homeostasis.Untargeted metabolomics analysis found that cornuside may be through Tryptophan metabolism,Taurine and hypotaurine metabolism,Primary bile acid biosynthesis and other metabolic pathways.Increased Xanthurenic acid,Pentadecanoic acid,Ferulic acid and other metabolites,Reducing the levels of metabolites such as Tauroursodeoxycholic acid,Oxoglutaric acid,and Imidazolepropionic acid to play a role.Through the correlation analysis of gut microbiota and metabolomics,it was found that cornuside could alleviate metabolic damage in diabetic mice mainly by increasing the abundance of Akkermansia,enhancing the Tryptophan metabolism pathway and up-regulating the level of Xanthurenic acid.In conclusion,cornuside could improve glucose metabolism in IR-HepG2 cells.It can improve glucose tolerance,insulin resistance,liver tissue damage and lipid accumulation,and reduce fasting blood glucose in T2DM mice.In addition,cornuside can alleviate the symptoms of diabetic mice by regulating the proportion of cecal flora and the level of metabolites,increasing the abundance of Akkermansia,enhancing the Tryptophan metabolism pathway and up-regulating the level of Xanthurenic acid.更多还原