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摘要
Objective To investigate the effect and molecular mechanism of triptolide on cluster of differentiation 36 (CD36) protein expression and lipid metabolism of testicular Sertoli cells. Methods Mouse testis Sertoli cell line TM4 cells were cultured in vitro. The survival rate of TM4 cells treated with triptolide (40, 80, 160, 320, 640 nmol·L?1) for 24 h was determined by CCK-8 method. The apoptosis rate of triptolide treated with 80, 160, 320 nmol·L?1 for 24 h was determined by flow cytometry. The accumulation of intracellular lipid droplets was detected by BODIPY staining and oil-red O staining after treated with triptolide (80, 160, 320 nmol·L?1) for 24 h. The level of triacylglycerol (TG) in cells treated with triptolide (80, 160, 320 nmol·L?1) for 24 h was detected by kit method. TM4 cells were pretreated with 0.1 mmol·L?1 adenosine triphosphate (ATP) for 3 h, and then treated with 160 nmol·L?1 triptolide for 24 h. The survival rate of TM4 cells was determined by CCK-8 method. Western blotting was used to detect the expression of CD36 protein in TM4 cells treated with triptolide (40, 80, 160, 320 nmol·L?1) for 24 h or 160 nmol·L?1 for 6, 12, 24, 36, 48 h. Western blotting was used to detect the protein expression levels of protein kinase 1 (PKD1) and its phosphorylated protein, histone deacetylase 7 (HDAC7) and forkhead box protein O1 (FOXO1) treated by triptolide (40, 80, 160, 320 nmol·L?1) for 24 h. Results Compared with control group, TM4 cell survival rate was significantly decreased in triptolide 80, 160, 320 and 640 nmol·L?1 groups (P < 0.05, 0.01), and IC50 was 214.1 nmol·L?1. The apoptosis rates of triptolide at 80, 160 and 320 nmol·L?1 were 11.89%, 23.17% and 32.12%, respectively, significantly different compared with control group (P < 0.05, 0.01). BODIPY staining showed that the intracellular red fluorescence intensity in triptolide group was significantly decreased compared with control group (P < 0.01). Oil red O staining also showed that the number of intracellular lipid droplets in triptolide 160 nmol·L?1 group was significantly lower than control group. Compared with control group, TG level in TM4 cells in triptolide group was significantly decreased (P < 0.01). Compared with triptolide group, ATP synergistic administration significantly reduced the inhibitory effect of triptolide on TM4 cell survival rate (P < 0.01). Compared with control group, the expression of CD36 protein in TM4 cells increased significantly after treated with triptolide at 80, 160 and 320 nmol·L?1 for 24 h (P < 0.01). The expression of CD36 protein in TM4 cells treated with triptolide at 160 nmol·L?1 for 6, 12, 24, 36 and 48 h. The expression level of CD36 first increased and then decreased with time, and reached the peak at 24 h (P < 0.05). Compared with control group, the levels of PKD1 and Ser 744-748 phosphorylation PKD1, protein expressions of HDAC7 and FOXO1 were significantly inhibited in TM4 cells in triptolide group (P < 0.05, 0.01). Conclusion Triptolide had obvious damage to testicular Sertoli cells. The mechanism of damage was related to the disturbance of lipid metabolism and ATP deficiency. The expression level of CD36 first increased and then decreased during injury, and the initial compensatory increase may be a stress protective mechanism of cells. Inhibition of PKD1/HDAC7/FOXO1 signaling pathway mediates the regulation of CD36 expression. ? 2022 The authors.
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