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-1 Cela 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-1 and 500 nmol·L-1.Oil red O staining and immunofluorescence results showed that 250 nmol·L-1 Cela significantly inhibited the adipogenic differentiation of 3T3-L1,250 nmol·L-1 Cela at the early stage(the first day)significantly inhibited the adipogenic differentiation of 3T3-L1.WB results showed that treatment with 250 nmol·L-1 celastrol 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-1 Cela,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-1 Cela can inhibit differentiation induced by 5121 inhibitor.Conclusion 250 nmol·L-1 Cela 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.
关键词
雷公藤红素/前体脂肪细胞/成脂分化/脂代谢/油红O染色/mmu-miR-5121
Key words
celastrol/3T3-L1/adipogenic differentiation/lipid metabolism/oil red O staining/mmu-miR-5121
维普
万方数据