白虎加人参汤对热应激下2 型糖尿病小鼠棕色脂肪组织AMPK 信号通路的影响研究

Study on the Effects of Baihu Plus Renshen Decoction on the AMPK Signaling Pathway in Brown Adipose Tissue of Type 2 Diabetic Mellitus Mice Under Heat Stress

摘要:

目的：探讨白虎加人参汤改善热应激下2型糖尿病（T2DM） 小鼠糖脂代谢紊乱的作用机制。方法：将36只4～5周龄SPF级雄性C57BL/6J小鼠按随机数字表法分为正常组（N组） 9只、模型组27只。N组普通饲料喂养；模型组高脂饲料喂养8周，8周后注射链脲佐菌素（STZ） 诱导T2DM模型，模型制备成功后按随机数字表法分为糖尿病热应激组（DMHS组）、白虎加人参汤组（BJRD组） 和二甲双胍组（MET组），每组9只。在注射STZ溶液造模的同时分组给药，BJRD组给予白虎加人参汤，MET组给予二甲双胍，N组和DMHS组给予生理盐水，共治疗4周。4周后除N组外，其余3组进行热应激实验。记录各组小鼠体质量变化；检测各组小鼠空腹血糖、空腹胰岛素水平，计算糖代谢指标；检测各组小鼠血脂水平；观察各组小鼠棕色脂肪组织（BAT） 的外观形态，HE染色观察病理改变，电镜下观察BAT超微结构改变；实时荧光定量-聚合酶链式反应法和蛋白质印迹法分别检测BAT中解偶联蛋白1（UCP1）、腺苷酸活化蛋白激酶（AMPK）、NAD-依赖性去乙酰化酶（SIRT1）、过氧化物酶体增殖物激活受体γ共激活因子1α（PGC1α） 的mRNA和蛋白表达水平。结果：与N组比较，DMHS组小鼠空腹血糖、空腹胰岛素水平、胰岛素抵抗指数均升高，胰岛素敏感指数和胰岛β细胞指数降低，差异均有统计学意义（P＜0.05）。与DMHS组比较，BJRD组小鼠的空腹血糖、空腹胰岛素水平、胰岛素抵抗指数均下降，胰岛素敏感指数和胰岛β细胞指数上升，差异均有统计学意义（P＜0.05）。与DMHS组比较，MET组的空腹胰岛素水平、胰岛素抵抗指数均下降（P＜0.05）。与N组比较，DMHS组的血清TC、TG、LDL-C含量上升（P＜0.05）。与DMHS组比较，BJRD组的血清TC、TG、LDL-C含量下降，HDL-C含量上升，差异均有统计学意义（P＜0.05）。与DMHS组比较，MET组血清TC、TG含量下降，HDL-C含量上升，差异均有统计学意义（P＜0.05）。与N组比较，DMHS组小鼠BAT质量、体质量及脏器指数均下降（P＜0.05）。HE染色结果提示白虎加人参汤可减轻热应激下T2DM小鼠的BAT白色化。电镜结果提示白虎加人参汤可减轻热应激下T2DM小鼠的BAT线粒体损伤。与N组比较，DMHS组小鼠BAT的UCP1、AMPK、SIRT1和PGC1α mRNA 表达水平均下降（P＜0.05）；与DMHS 组比较，BJRD 组小鼠BAT 的UCP1、AMPK、SIRT1 和PGC1α mRNA表达水平均升高（P＜0.05）；与DMHS组比较，MET组小鼠BAT的SIRT1和PGC1α mRNA表达水平升高（P＜0.05）。与N组比较，DMHS组小鼠BAT的UCP1、p-AMPK/AMPK、SIRT1和PGC1α蛋白表达水平均下降（P＜0.05）；与DMHS组比较，BJRD组小鼠BAT的UCP1、p-AMPK/AMPK、SIRT1和PGC1α蛋白表达水平均升高（P＜0.05）；与DMHS组比较，MET组小鼠BAT的p-AMPK/AMPK和PGC1α蛋白表达水平均升高（P＜0.05）。结论：白虎加人参汤可能通过激活热应激下T2DM小鼠BAT中的AMPK-SIRT1-PGC1α通路进而增强BAT的活性，以改善热应激下T2DM小鼠的糖脂代谢紊乱。

Abstract:

Abstract： Objective： To investigate the mechanism of action of Baihu Plus Renshen Decoction （BJRD） in improving glucolipid metabolic disorders in type 2 diabetes mellitus （T2DM） mice under heat stress. Methods：A total of 36 male SPF-grade C57BL/6J mice aged 4-5 weeks were randomly divided into a normal group （N group，n=9） and a model group（ n=27） using a random number table method. Mice in the N group were fed a normal diet，while those in the model group were fed a high-fat diet for 8 weeks，followed by intraperitoneal injection of streptozotocin （STZ） to induce T2DM. After successful modeling， the model group was further randomly divided into three subgroups： the diabetes mellitus with heat stress group （DMHS group），the Baihu Plus Renshen Decoction group （BJRD group），and the metformin group （MET group），with 9 mice in each group. Drug administration was initiated simultaneously with STZ injection. The BJRD group received Baihu Plus Renshen Decoction，the MET group received metformin，and the N and DMHS groups received normal saline，all for 4 weeks. After the 4-week treatment，all groups except the N group were subjected to heat stress experiments. Body weight changes were recorded. Fasting blood glucose and fasting insulin levels were measured，and glycemic metabolic indicators were calculated. Levels of blood lipid indicators were assessed. The gross morphology of brown adipose tissue （BAT） was observed； histopathological changes were evaluated by hematoxylin-eosin （HE） staining； and ultrastructural changes in BAT were examined by electron microscopy. The mRNA and protein expression levels of uncoupling protein 1（ UCP1），AMP-activated protein kinase（ AMPK），NADdependent deacetylase sirtuin-1 （ SIRT1）， and peroxisome proliferator-activated receptor gamma coactivator 1-alpha （PGC1α） in BAT were detected by real-time quantitative polymerase chain reaction （RT-qPCR） and Western blotting，respectively. Results：Compared with the N group，the DMHS group showed significantly increased fasting blood glucose levels， fasting insulin levels， and homeostatic model assessment of insulin resistance （HOMA-IR）， along with decreased insulin sensitivity index （ISI） and homeostatic model assessment of β-cell function （HOMA-β）， with all differences reaching statistical significance （P＜0.05）. Compared with the DMHS group， the BJRD group exhibited significantly decreased fasting blood glucose，fasting insulin levels，and HOMA-IR，as well as increased ISI and HOMA-β（ P＜0.05）. Compared with the DMHS group，the MET group showed decreased fasting insulin levels and HOMA-IR （P＜0.05）. Compared with the N group， the DMHS group showed significantly increased serum levels of total cholesterol （TC），triglycerides （TG），and low-density lipoprotein cholesterol （LDL-C） （P＜0.05）. Compared with the DMHS group，the BJRD group showed significantly decreased serum TC，TG，and LDL-C levels，along with increased high-density lipoprotein cholesterol （HDL-C） levels （P＜0.05）. The MET group also showed significantly decreased TC and TG levels and increased HDL-C levels compared with the DMHS group （P＜0.05）. BAT weight， body weight，and organ index were significantly decreased in the DMHS group compared with the N group （P＜0.05）. HE staining indicated that BJRD alleviated BAT whitening in T2DM mice under heat stress. Electron microscopy results suggested that BJRD mitigated mitochondrial damage in BAT of T2DM mice under heat stress. Compared with the N group，the mRNA expression levels of UCP1，AMPK，SIRT1，and PGC1α in BAT were significantly decreased in the DMHS group （P＜0.05）. Compared with the DMHS group， the BJRD group showed significantly increased mRNA expression levels of UCP1， AMPK， SIRT1， and PGC1α in BAT （P＜0.05）. Compared with the DMHS group， the MET group showed significantly increased mRNA expression levels of SIRT1 and PGC1α（ P＜0.05）. Compared with the N group， the protein expression levels of UCP1， p-AMPK/AMPK， SIRT1， and PGC1α in BAT were significantly decreased in the DMHS group （P＜0.05）. Compared with the DMHS group， the BJRD group showed significantly increased protein expression levels of UCP1， p-AMPK/AMPK， SIRT1， and PGC1α （P＜0.05）. Compared with the DMHS group， the MET group showed significantly increased protein expression levels of p-AMPK/AMPK and PGC1α （P＜0.05）. Conclusion： BJRD may enhance the activity of BAT by activating the AMPK-SIRT1-PGC1α pathway in BAT，thus improving glucose and lipid metabolic disorders in T2DM mice under heat stress.