Untargeted Metabolomics Investigating the Intracellular Metabolic Alterations of HaCaT Cells Treated with Isotretinoin

Shengcai Zhu,1 Yang He,1 Liang Wu,1 Xiaoliang Ouyang,2 Quan Wei,1 Fan Ye,1 Chunming Li1 1Department of Dermatology, The Second Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang, Jiangxi, People’s Republic of China; 2Department of Plastic Surgery, The Second Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang, Jiangxi, People’s Republic of ChinaCorrespondence: Chunming Li, Department of Dermatology, The Second Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang, Jiangxi, People’s Republic of China, Email [email protected]: Isotretinoin has been increasingly utilized for various dermatological diseases.However, the mechanism of action of isotretinoin on the skin, particularly the epidermis, remains unclear.Purpose: This study aimed to investigate the intracellular metabolic alterations induced by isotretinoin in HaCaT cells.Methods: HaCaT cells were divided into three groups: Group A (control group), Group B(treated with 10 μM isotretinoin for 48 h), and Group C (treated with 10 μM isotretinoin for 7 d). An untargeted metabolomics analysis was employed using ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-Q-TOF/MS). Multivariate data analyses were employed to identify distinguishing metabolites, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) was utilized to assess potential metabolic pathways.Results: Isotretinoin inhibited the proliferation of HaCaT cells in a dose-dependent manner. The OPLS-DA model displayed clear segmentation between the Group B and Group A, as well as between Group C and Group A. Compared to Group A, there were 115 significantly differentially metabolites in Group B, including glycerophosphocholine,adenosine,acetylcarnitine,creatine,Dl-lactate,hypoxanthine,uracil,LPC 18:1,creatine phosphate,5′-S-Methyl-5′-thioadenosine. In Group C, there were 140 significantly differentially expressed metabolites compared to Group A, including glycerophosphocholine, niacinamide, creatine, phytosphingosine, oleic acid, hypoxanthine, 1-Palmitoyl-sn-glycero-3-phosphocholine, Dl-lactate, 5′-S-Methyl-5′-thioadenosine and NG,NG-Dimethyl-L-arginine.A total of 30 metabolic pathways were significantly changed (p < 0.05) in Group A and Group B.The most significant metabolic pathways included purine metabolism,nucleotide metabolism,ABC transporters, and the taurine and hypotaurine metabolism signaling pathway. Additionally, 46 metabolic pathways were significantly altered (p < 0.05) in Group A and Group C, with the most significant pathways being nucleotide metabolism, ABC transporters, purine metabolism, and glycerophospholipid metabolism.Conclusion: Important metabolites and metabolic pathways were identified in this study,which will help clarify the underlying mechanism of action of isotretinoin on keratinocytes at the metabolite level and the mucocutaneous adverse effects associated with isotretinoin in the treatment of dermatological diseases.Keywords: Isotretinoin, metabolomics, HaCaT cells, purine metabolism