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Background: Jian-Gan-Xiao-Zhi decoction (JGXZ) is a traditional Chinese medicine formula to treat patients with non-alcoholic fatty liver disease (NAFLD). The study aimed to analyze the mechanism of JGXZ in adipocytes and detect the main components of the drug in rat serum. Methods: 3T3-L1 preadipocytes were used to establish an insulin resistant (IR) adipocyte model. Lipid accumulation in adipocytes was detected by oil red O staining. After JGXZ treatment, glucose consumption, total cholesterol (TC), and triglyceride (TG) were analyzed using the corresponding kits. ROS levels were measured by flow cytometry. In addition, Western blot was used to assess LKB1/AMPK and JNK/IRS/PI3k/AKT expressions. The main components of JGXZ in rat serum samples were detected by LC-MS/MS using a Phenomenex Luna C18 column, a mobile phase of methanol and 0.1% formic acid solution, and ESI detection. Results: JGXZ significantly decreased glucose levels and adipogenesis, accompanied by decreased IR (P < 0.01). Besides, JGXZ markedly affected ROS, LKB1/AMPK, and JNK/IRS/PI3k/AKT levels (P < 0.01). R1, Rg1, paeoniflorin, Rb1, astragaloside IV, and tanshinone could be significantly quantified. Conclusions: JGXZ decreased glucose and lipid synthesis, possibly via the ROS/AMPK/JNK pathway. R1, Rg1, paeoniflorin, Rb1, astragaloside IV, and tanshinone in JGXZ could play major roles in treating NAFLD, which could assist in the study of the mechanism of JGXZ in treating NAFLD.