The inhibitory effect and mechanism of Celastrol on adipogenic differentiation in 3T3-L1

doi:10.19803/j.1672-8629.20230708

Abstract

Abstract: Objective To study the inhibitory effect and mechanism of Celastrol (Cela) on adipogenic differentiation in preadipocytes (3T3-L1). Methods CCK-8 was used to determine the optimal concentration of Cela for 3T3-L1 cells. Oil red O staining and immunofluorescence (Perilipin1, Adiponectin) were used to imply the inhibitory effect of Celastrol on 3T3-L1 adipogenic differentiation at different stages. Western Blot (WB) was used to detect proteins expression related to lipid metabolism and differentiation in 3T3-L1 cells treated with different concentrations of Cela; WB, microRNA (miRNA) sequencing and qPCR were used to analyze undifferentiated (Con), differentiation Day1 (MDI), 250 nmol·L^-1Cela treatment on the first day of differentiation (MCT), and KEGG and GO enrichment analysis were performed; mmu-miR-5121 mimics and inhibitor were overexpressed, then changes in related proteins were detected by WB, and differentiation results were detected by oil red O staining. Results The optimal concentrations of celastrol for treatment of 3T3-L1 cells were 250 nmol·L^-1and 500 nmol·L^-1. Oil red O staining and immunofluorescence results showed that 250 nmol·L^-1Cela significantly inhibited the adipogenic differentiation of 3T3-L1, 250 nmol·L^-1Cela at the early stage (the first day) significantly inhibited the adipogenic differentiation of 3T3-L1. WB results showed that treatment with 250 nmol·L^-1celastrol significantly inhibited the protein expression of DFCP1, FAS, ACC, and PPARγ in 3T3-L1 cells. On the first day of differentiation, the expression of FAS and ACC increased. After treatment with 250 nmol·L^-1Cela, the expression of ACC and FAS protein significantly decreased; miRNA sequencing screening and validation showed that mmu-miR-5121 decreased on the first day of differentiation, but increased after Cela treatment. Transfecting 5121 inhibitor increased the content of FAS and ACC, while 5121 mimics showed the opposite effect. Transfecting 5121 inhibitor alone led to adipogenic differentiation, while 5121 mimics could effectively inhibit normal differentiation. And 250 nmol·L^-1Cela can inhibit differentiation induced by 5121 inhibitor. Conclusion 250 nmol·L^-1Cela significantly inhibited the differentiation at early state of 3T3-L1 adipogenic differentiation, possibly by upregulating the content of mmu-miR-5121 and inhibiting the expression of FAS and ACC.

Key words: celastrol, 3T3-L1, adipogenic differentiation, lipid metabolism, oil red O staining, mmu-miR-5121

CLC Number:

R977
R994.1

YUE Lanxin, SHEN Pan, ZHOU Lei, HUANG Congshu, ZHOU Wei, GAO Yue. The inhibitory effect and mechanism of Celastrol on adipogenic differentiation in 3T3-L1[J]. Chinese Journal of Pharmacovigilance, 2024, 21(1): 65-73.

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