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Proteomic screening identifies the direct targets of chrysin anti-lipid depot in adipocytes

作者 Mei, Jie; Yang, Rong; Yang, Qiaohong; Wan, Wencheng; Wei, Xiaoyong

期刊 JOURNAL OF ETHNOPHARMACOLOGY , MAR 1 2021,:267

DOI 10.1016/j.jep.2020.113361 PubMed 32891819

摘要 Ethnopharmacological relevance: Overweight/obesity was mentioned by many countries as an obstacle to good health and long life, which increases risk of diseases and disorders. Previous studies suggested that the chronic low-grade inflammation present in the body was considered as the essential pathogenesis for obesity. Chrysin is extracted from traditional Chinese medicine Oroxylum indicum (Linn.) Kurz and plays a superior anti-obesity role. Chrysin could reduce the lipid depot by inhibiting the obesity-related inflammation in adipose tissue. However, the target protein for chrysin to exert its anti-obesity role are not verified.Aim of study: The present study aimed to screen and validate the target protein for chrysin to reduce the lipid depot in palmitic acid-induced 3T3-L1 adipocytes.Materials and methods: Obesity model was established employing 0.5 mmol/L palmitic acid-induced 3T3-L1 adipocytes through Cocktails method. Two-dimensional gel electrophoresis (2-DE) combined with liquid chromatography-mass spectrometry (LC-MS) was applied to analyze the differentially expressed proteins for chrysin intervention by lipid formation in adipocytes. Gene silencing was utilized to decrease gene expression of the candidate proteins, then production of triglyceride in 3T3-L1 was detected by triglycerides assay to determine the target proteins. Ultraviolet (UV) absorption together with fluorescence spectra validated the direct target proteins of chrysin. They also computed the correlation constants of combination between chrysin and the target proteins. Molecular docking was further employed to identify the main binding amino acids between chrysin and the target protein.Results: 2-DE combined with LC-MS screened four candidate proteins which were related to metabolism and inflammation. The production of triglycerides in 3T3-L1 was reduced after decreasing gene expression of Annexin A2 (ANXA2), 60 kDa heat shock protein (HSP-60) and succinyl-CoA:3-ketoacid coenzyme A transferase 1 (SCOT-S), respectively. UV spectrum showed that the absorbance spectra of ANXA2 from 260 to 300 nm shifted upwards along with the increase in chrysin concentration, meanwhile the absorbance spectra of HSP-60 from 200 to 220 nm and from 265 to 280 nm shifted slightly upwards along with the increase in chrysin concentrations. The results indicated the conjugated structures between chrysin and ANXA2 or HSP-60. Fluorescence quenching further suggested a spontaneous interaction between chrysin and ANXA2 or HSP-60. Finally, molecular docking identified the main binding amino acids between ANXA2 and chrysin were Ser22, Tyr24, Pro267, Val298, Asp299, and Lys302.Conclusions: Chrysin can reduce the amount of triglycerides by directly downregulating the inflammation-related target proteins ANXA2 and HSP-60, exerting an anti-obesity role.

涉及物种 千张纸 Oroxylum indicum