Phytomedicine : international journal of phytotherapy and phytopharmacology · 2025
Celastrol attenuates Th17 cell response through downregulating HDAC3/FOXO3/GLS1-dependent glutaminolysis.
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Liu Yan, Zhang Wenjie, Guo Yilei, Wei Zhifeng, Xia Yufeng, Dai Yue
Аннотация
BACKGROUND: Preparations of Tripterygium wilfordii have long been used in China for the treatment of various autoimmune diseases. Celastrol, one of the primary active ingredients of T. wilfordii, was previously reported to attenuate the differentiation of Th17 cells which are closely related to the pathogenesis of autoimmune diseases, but its underlying mechanism remains to be identified.
PURPOSE: This study investigated the effect of celastrol on Th17 cell response and explored the mechanism of action based on cellular metabolism.
METHODS: Primary CD4+ T cells isolated from the mesenteric lymph nodes of C57BL/6 mice were stimulated with Th17 cell differentiation conditions. The Th17 cell proportion, mRNA expression of key transcription factor and cytokines, protein level of histone deacetylases (HDACs) as well as concentration of glutamine and its metabolites were detected by flow cytometry, real-time quantitative PCR (RT-qPCR), western blot and ELISA, respectively. The transcription factor for glutaminase 1 (GLS1) was predicted by database and verified by ChIP-qPCR and luciferase reporter assay. The binding of celastrol to HDAC was simulated by molecular docking and further verified by protein thermal stability detection, microscale thermophoresis and siRNA transfection. Colitis model was established in mice using dextran sulfate sodium (DSS).
RESULTS: In vitro, celastrol inhibited Th17 cell differentiation in a concentration-dependent manner. Metabolic screening revealed that celastrol preferentially downregulated glutaminolysis, as evidenced by increased intracellular glutamine levels and decreased GLS1 expression at both protein and mRNA levels. Exogenous supplement of glutamine or glutaminolysis products reversed the effect of celastrol, indicating that celastrol attenuates Th17 differentiation by inhibiting GLS1-dependent glutaminolysis. Database screening identified forkhead box 3 (FOXO3) as a key transcription factor regulating GLS1 expression. Celastrol interacted with HDAC3, reducing its activity and leading to increased acetylation and upregulation of FOXO3 expression. FOXO3, in turn, bound to the GLS1 promoter, resulting in downregulation of GLS1 expression. In mice with DSS-induced colitis, the inhibitory effect of celastrol on Th17 cell response was verified.
CONCLUSION: Celastrol mitigates the Th17 cell response by downregulating HDAC3/FOXO3/GLS-dependent glutaminolysis, highlighting its potential as a therapeutic agent for Th17 cell activation-related autoimmune diseases.
PURPOSE: This study investigated the effect of celastrol on Th17 cell response and explored the mechanism of action based on cellular metabolism.
METHODS: Primary CD4+ T cells isolated from the mesenteric lymph nodes of C57BL/6 mice were stimulated with Th17 cell differentiation conditions. The Th17 cell proportion, mRNA expression of key transcription factor and cytokines, protein level of histone deacetylases (HDACs) as well as concentration of glutamine and its metabolites were detected by flow cytometry, real-time quantitative PCR (RT-qPCR), western blot and ELISA, respectively. The transcription factor for glutaminase 1 (GLS1) was predicted by database and verified by ChIP-qPCR and luciferase reporter assay. The binding of celastrol to HDAC was simulated by molecular docking and further verified by protein thermal stability detection, microscale thermophoresis and siRNA transfection. Colitis model was established in mice using dextran sulfate sodium (DSS).
RESULTS: In vitro, celastrol inhibited Th17 cell differentiation in a concentration-dependent manner. Metabolic screening revealed that celastrol preferentially downregulated glutaminolysis, as evidenced by increased intracellular glutamine levels and decreased GLS1 expression at both protein and mRNA levels. Exogenous supplement of glutamine or glutaminolysis products reversed the effect of celastrol, indicating that celastrol attenuates Th17 differentiation by inhibiting GLS1-dependent glutaminolysis. Database screening identified forkhead box 3 (FOXO3) as a key transcription factor regulating GLS1 expression. Celastrol interacted with HDAC3, reducing its activity and leading to increased acetylation and upregulation of FOXO3 expression. FOXO3, in turn, bound to the GLS1 promoter, resulting in downregulation of GLS1 expression. In mice with DSS-induced colitis, the inhibitory effect of celastrol on Th17 cell response was verified.
CONCLUSION: Celastrol mitigates the Th17 cell response by downregulating HDAC3/FOXO3/GLS-dependent glutaminolysis, highlighting its potential as a therapeutic agent for Th17 cell activation-related autoimmune diseases.