20S-原人参三醇改善糖尿病肾病的作用研究

Effect of 20S-Protopanaxatriol on diabetic kidney disease

关键词:  糖尿病肾病  20S-原人参三醇  质谱成像  药效学评价

Key words:  diabetic kidney disease  20S-Protopanaxatriol  mass spectrometry imaging  pharmacodynamic evaluation

摘要:

[目的] 探讨人参中活性物质20S-原人参三醇（20S-PPT）在糖尿病肾病（DKD）发生发展过程中的治疗作用。[方法] 采用高糖诱导的人肾小管上皮细胞（HK-2）损伤，模拟DKD的体外模型。设置对照组、高糖组、20S-PPT组，孵育48 h后，通过细胞形态、数量、活力、凋亡情况、线粒体功能等指标的检测，初步评估20S-PPT对DKD的潜在治疗作用。从动物模型探讨药物疗效方面出发，建立DKD小鼠模型后随机分为正常组、模型组和2OS-PPT组。其中，2OS-PPT组通过灌胃的方式获得20S-PPT，给药4周。随后，通过观察分析小鼠的一般指标，检测肾组织病理生化指标、氧化应激指标，定性定量的评估20S-PPT对小鼠肾损伤的药物疗效。并通过质谱成像技术，探究20S-PPT对DKD进展中代谢紊乱的影响。[结果] 与对照组相比，高糖（50 mmol/L）组处理48 h时，HK-2细胞形态从鹅卵石状转化为纤维状、细胞数量减少（P ＜0.01）、活力降低（P ＜0.01）、凋亡率及活性氧（ROS）水平升高（P ＜0.05）、线粒体膜电位下降；与高糖组相比，20S-PPT（5 μmol/L）组处理48 h时，HK-2细胞形态向鹅卵石状转变、细胞数量及活力发生回调（P ＜0.05）、细胞凋亡率及ROS水平降低（P ＜0.05）、线粒体膜电位升高。与正常组相比，模型组小鼠空腹血糖（FBG）、尿白蛋白/肌酐比值（UACR）、口服糖耐量（OGTT）、肾指数显著升高（P ＜0.05），肌酐（CRE）、尿素氮（BUN）、尿酸（UA）、总胆固醇（TC）、三酰甘油（TG）、低密度脂蛋白（LDL-C）及丙二醛（MDA）水平显著升高（P ＜0.05），高密度脂蛋白（HDL-C）及超氧化物歧化酶（SOD）和谷胱甘肽过氧化物酶（GSH-Px）水平明显降低（P ＜0.05）；与模型组相比，20S-PPT（20 mg/kg）组小鼠FBG、OGTT、肾指数显著降低（P ＜0.05），CRE、BUN、UA、TC、TG、LDL-C及MDA水平显著降低（P ＜0.05），HDL-C及SOD和GSH-Px水平明显升高（P ＜0.05）。此外，20S-PPT能够调节DKD小鼠体内多种代谢通路，如核苷酸代谢、糖脂代谢、三羧酸循环。[结论] 体外研究表明20S-PPT在5 μmol/L浓度下，给药48 h时可通过调控细胞凋亡、氧化应激等作用，改善高糖状态下HK-2细胞损伤；动物实验进一步证实20S-PPT（20 mg/kg）可调节DKD小鼠肾功能及病理损伤；其具体的作用机制与代谢调控密切相关。

Abstract:

[Objective] To explore the therapeutic effect of 20S-Protopanaxatriol（20S-PPT），an active substance in ginseng，on the occurrence and development of diabetic kidney disease（DKD）. [Method] Using high glucose induced injury of human renal tubular epithelial cells（HK-2） to simulate an in vitro model of DKD. Set up control group，high glucose group，high glucose+20S-PPT group. After 48 hours of incubation，the potential therapeutic effect of 20S-PPT on DKD was preliminarily evaluated by detecting cell morphology，quantity，viability，apoptosis，mitochondrial function，and other indicators. Next，starting from the exploration of drug efficacy in animal models， a DKD mouse models were established and randomly divided into normal group，model group and 20S-PPT group. The 20S-PPT group obtained 20S-PPT by gavage and received treatment for 4 weeks. Subsequently，by observing and analyzing the general indicators of mice，detecting renal tissue pathological and biochemical indicators，oxidative stress indicators，and qualitatively and quantitatively evaluating the drug efficacy of 20S-PPT on mouse kidney injury. And explore the effect of 20S-PPT on metabolic disorders in the progression of DKD through mass spectrometry imaging technology. [Result] Compared with the control group，after 48 hours of treatment with high glucose（50 mmol/L），the morphology of HK-2 cells transformed from pebble shaped to fibrous，the number of cells decreased（P<0.01），the vitality decreased（P<0.01），the apoptosis rate and reactive oxygen species（ROS） level increased（P<0.05），and the mitochondrial membrane potential decreased；Compared with the high glucose group，the 20S-PPT（5 μmol/L） group showed a pebble shaped transformation in HK-2 cell morphology，a decrease in cell number and vitality（P<0.05），a decrease in cell apoptosis rate and ROS levels（P<0.05），and an increase in mitochondrial membrane potential after 48 hours of treatment. Compared with the control group，the fasting blood glucose，urinary albumin creatinine ratio，oral glucose tolerance，and renal index of the DKD model group mice were significantly increased（P<0.05）. The levels of creatinine，urea nitrogen，uric acid，total cholesterol，triglycerides，low-density lipoprotein cholesterol，and malondialdehyde were significantly increased（P<0.05），while the levels of high-density lipoprotein cholesterol，superoxide dismutase，and glutathione peroxidase were significantly decreased（P<0.05）. Compared with the DKD model group，the mice in the 20S-PPT（20 mg/kg） administration group showed significant reductions in fasting blood glucose，oral glucose tolerance，and renal index（P<0.05）. The levels of creatinine， urea nitrogen，uric acid，total cholesterol，triglycerides，low-density lipoprotein cholesterol，and malondialdehyde were significantly reduced（P<0.05），while the levels of high-density lipoprotein cholesterol，superoxide dismutase，and glutathione peroxidase were significantly increased（P<0.05）. In addition，20S-PPT can regulate various metabolic pathways in DKD mice，such as nucleotide metabolism， glucose and lipid metabolism，and tricarboxylic acid cycle. [Conclusion] In vitro studies have shown that 20S-PPT can improve HK-2 cell damage under high glucose conditions by regulating cell apoptosis，oxidative stress，and other effects at a concentration of 5 μmol/L after 48 hours of administration. Animal experiments further confirmed that 20S-PPT（20 mg/kg） can regulate renal function and pathological damage in DKD mice. Its specific mechanism of action is closely related to metabolic regulation.