The research focuses on the influence of electroacupuncture (EA) on hepatic autophagy mechanisms in obese mice, particularly its modulation via the AMPK/mTOR/ULK1 pathway. By examining this impact, the study aims to shed light on potential therapeutic avenues for obesity-related liver dysfunction.
In the study, C57BL/6J mice were utilized to develop an obesity model through a high-fat diet. The experimental subjects were divided into three groups: a normal group, a model group, and an EA-treated group, with eight mice in each category. The EA treatment employed stimulation at bilateral Zusanli (ST36) and Tianshu (ST25) acupuncture points, executed for 30 minutes, five times a week, over the span of four weeks.
To assess the effects of the treatments, various metrics were monitored both before and after the intervention. The assessment included Lee's index, body mass measurements, and fasting serum glucose levels. Additionally, an insulin tolerance test (ITT) was conducted to evaluate insulin sensitivity, with the area under the curve (AUC) calculated for analysis.
A suite of biochemical assays was performed using enzyme-linked immunosorbent assay (ELISA) to measure levels of fasting insulin, serum low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), total cholesterol (TC), and triacylglycerol (TG). Furthermore, the study calculated the insulin resistance index and evaluated the mass of mesenteric white adipose tissue (mWAT) alongside liver tissue. Histological examinations of the liver and mWAT were performed using HE staining, and lipid deposition was observed via Oil Red O staining. The ultrastructure of liver tissue was further evaluated using transmission electron microscopy.
Protein expression levels of p-AMPK/AMPK, p-mTOR/mTOR, ULK1, and autophagy markers such as LC3-Ⅱ, Atg5, Atg7, and p62 were analyzed through Western blotting. Additionally, the mRNA expressions of these proteins were quantified via real-time PCR.
The findings indicated several significant alterations when comparing the model group to the normal group. The model group presented significant increases in Lee's index, body mass, fasting blood glucose, fasting insulin, insulin resistance index, and ITT-AUC (P < 0.01). Additionally, serum levels of TG, TC, and LDL-C were markedly elevated (P < 0.01), while the HDL-C levels exhibited a significant decrease (P < 0.01).
Morphologically, the model group exhibited increased mass in both mWAT and liver tissues (P < 0.01), enlarged adipocytes with lower cell count per unit area in mWAT, swollen hepatocytes with numerous lipid droplets and vacuoles, and a pronounced orange-red lipid deposition. The ultrastructural analysis revealed mitochondrial shrinkage and sparse autophagic vacuoles.
At the molecular level, the model group experienced decreased expressions of AMPK mRNA and p-AMPK/AMPK protein (P < 0.01) alongside increased mTOR mRNA and p-mTOR/mTOR protein expression (P < 0.01). Notably, there were reductions in ULK1, LC3-Ⅱ, Atg5, and Atg7 mRNA and protein expressions (P < 0.01), coupled with elevated levels of p62 (P < 0.01).
In contrast, the EA group exhibited significant reductions in all the aforementioned parameters compared to the model group. The EA treatment led to decreases in Lee's index, body mass, fasting blood glucose, fasting insulin, and the insulin resistance index (P < 0.01). Furthermore, significant improvements in lipid profiles (decreased TG, TC, LDL-C, with increased HDL-C) were observed, indicating enhanced metabolic health post-treatment (P < 0.01).
Histological analysis revealed a reduction in adipocyte size and an increase in cell density in mWAT, along with normalized liver histology, characterized by diminished lipid droplets and vacuoles in hepatocytes. The ultrastructure of liver tissues appeared improved, showcasing restored mitochondrial integrity and increased autophagic vacuoles.
The results from this study suggest that electroacupuncture applied at ST36 and ST25 effectively promotes hepatic autophagy in obese mice, likely through the regulation of the AMPK/mTOR/ULK1 signaling pathway. This research underscores the potential of electroacupuncture as an innovative approach for treating obesity-related hepatic complications, warranting further investigation into its mechanistic role in metabolic regulation.