College of Pharmaceutical Sciences, Zhejiang Chinese Medical University, Hangzhou, 310053, China.
College of Basic Medicine, Zhejiang Chinese Medical University, Hangzhou, 310053, China.
J Ethnopharmacol. 2021 Apr 24;270:113828. doi: 10.1016/j.jep.2021.113828. Epub 2021 Jan 18.
Diabetes is a serious chronic metabolic disorder, and type 2 diabetes mellitus (T2DM) accounts for more than 90% of all diabetes cases. Insulin resistance (IR) is an early symptom, typical feature and main pathogenesis of T2DM due to the combined effects of genetic and environmental factors. Current evidence shows that IR is mainly caused by nutrient overload, systemic fatty acid excess, fatty tissue inflammation, endoplasmic reticulum stress, oxidative stress and abnormal autophagy. Autophagy plays an important role in the development of IR and decreased autophagy activity can cause IR through various ways.
Yunpiheluo (YPHL) decoction is a Chinese herbal formula with unique advantages for the treatment of T2DM. The aim of the present study was to investigate the regulatory mechanism of YPHL on the autophagy pathway in the skeletal muscle of IR Zucker diabetic fatty (ZDF) rats.
T2DM ZDF rats were treated with YPHL or transfected with SIRT1 adeno-associated virus. Serum total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), insulin resistance index (IRI) and skeletal muscle TG levels were detected in a T2DM ZDF rat model. The skeletal muscle morphology was observed by histological analysis and Oil Red O Staining. Autophagosomes were observed by transmission electron microscopy (TEM). The skeletal muscle morphology and fat deposition were observed by histological examination and Oil Red O Staining. A rat skeletal muscle IR cell model was established and transfected with SIRT1 overexpression plasmids. Cell apoptosis was observed by DAPI staining. SIRT1 levels in skeletal muscle tissues and cells were detected by qRT-PCR. The protein expressions of SIRT1, FOXo1, LC3B and P62 were detected by Western blotting.
Large numbers of lipid droplets and swollen mitochondria were observed in the skeletal muscle in both model group and negative control (NC) group receiving blank plasmid. Autophagosomes were seen in the skeletal muscle of YPHL and SIRT1 groups, with no significant structural abnormality. In addition, the protein expression of LC3B was decreased and the protein expression of p62 was increased significantly in the model group as compared with the NC group. After intervention with YPHL and SIRT1 overexpression, the protein expression of LC3B was significantly increased and p62 was significantly decreased. However, there was no significant difference in cell apoptosis between the two groups.
The SIRT1-FoxO1 autophagy pathway may play a significant role in the pathogenesis of IR. YPHL could increase the autophagy level by regulating the SIRT1-FoxO1 signaling pathway in the skeletal muscle and improving the lipid metabolism, thereby attenuating IR.
糖尿病是一种严重的慢性代谢紊乱，其中 2 型糖尿病（T2DM）占所有糖尿病病例的 90%以上。胰岛素抵抗（IR）是 T2DM 的早期症状、典型特征和主要发病机制，这是由于遗传和环境因素的综合作用。目前的证据表明，IR 主要是由营养物质过载、全身脂肪酸过多、脂肪组织炎症、内质网应激、氧化应激和异常自噬引起的。自噬在 IR 的发展中起着重要作用，自噬活性的降低可以通过多种方式引起 IR。
运脾活络（YPHL）汤是一种治疗 T2DM 的中药方剂，具有独特的优势。本研究旨在探讨 YPHL 对 IR Zucker 肥胖型（ZDF）糖尿病大鼠骨骼肌自噬途径的调节机制。
在 T2DM ZDF 大鼠模型中，用 YPHL 治疗或转染 SIRT1 腺相关病毒。检测 T2DM ZDF 大鼠血清总胆固醇（TC）、三酰甘油（TG）、高密度脂蛋白（HDL）、低密度脂蛋白（LDL）、胰岛素抵抗指数（IRI）和骨骼肌 TG 水平。通过组织学分析和油红 O 染色观察骨骼肌形态。通过透射电子显微镜（TEM）观察自噬体。通过组织学检查和油红 O 染色观察骨骼肌形态和脂肪沉积。建立大鼠骨骼肌 IR 细胞模型，并转染 SIRT1 过表达质粒。通过 DAPI 染色观察细胞凋亡。通过 qRT-PCR 检测骨骼肌组织和细胞中 SIRT1 水平。通过 Western blot 检测 SIRT1、FOXO1、LC3B 和 P62 的蛋白表达。
模型组和空载质粒阴性对照组（NC 组）骨骼肌中可见大量脂滴和肿胀的线粒体。YPHL 和 SIRT1 组骨骼肌中可见自噬体，未见明显结构异常。此外，与 NC 组相比，模型组 LC3B 蛋白表达降低，p62 蛋白表达显著升高。用 YPHL 和 SIRT1 过表达干预后，LC3B 蛋白表达显著增加，p62 蛋白表达显著降低。然而，两组细胞凋亡无明显差异。
SIRT1-FoxO1 自噬途径可能在 IR 的发病机制中起重要作用。YPHL 可通过调节骨骼肌中 SIRT1-FoxO1 信号通路，改善脂质代谢，从而增加自噬水平，减轻 IR。
