目的 研究雷公藤甲素影響睪丸支持細胞的人白細胞分化抗原36（CD36）蛋白表達及脂質(zhì)代謝的作用與分子機制。方法 體外培養(yǎng)小鼠睪丸支持細胞系TM4細胞，CCK-8法檢測雷公藤甲素（40、80、160、320、640 nmol·L^-1）作用24 h細胞存活率；流式細胞術(shù)測定雷公藤甲素（80、160、320 nmol·L^-1）作用24 h細胞凋亡率；氟硼二吡咯化合物（BODIPY）染色和油紅O染色檢測雷公藤甲素（80、160、320 nmol·L^-1）作用24 h細胞內(nèi)脂滴聚積情況；試劑盒法檢測雷公藤甲素（80、160、320 nmol·L^-1）作用24 h細胞內(nèi)三酰甘油（TG）水平；TM4細胞經(jīng)0.1 mmol·L^-1三磷酸腺苷（ATP）預(yù)處理3 h后，再與160 nmol·L^-1雷公藤甲素共處理24 h，用CCK-8法檢測TM4細胞的存活率；Western blotting法檢測雷公藤甲素（40、80、160、320 nmol·L^-1）作用24 h、或160 nmol·L^-1雷公藤甲素作用6、12、24、36、48 h后TM4細胞中CD36蛋白表達量；Western blotting法檢測雷公藤甲素（40、80、160、320 nmol·L^-1）作用24 h蛋白激酶1（PKD1）及其磷酸化蛋白、組蛋白去乙?；?（HDAC7）和叉頭框蛋白O1（FOXO1）的蛋白表達水平。結(jié)果 與對照組比較，雷公藤甲素80、160、320、640 nmol·L^-1濃度組TM4細胞存活率顯著下降（P<0.05、0.01），半數(shù)抑制濃度（IC₅₀）為214.1 nmol·L^-1； 80、160、320 nmol·L^-1雷公藤甲素的細胞凋亡率分別為11.89%、23.17%、32.12%，與對照組比較差異顯著（P<0.05、0.01）。BODIPY染色結(jié)果顯示，與對照組比較，雷公藤甲素組細胞內(nèi)的紅色熒光強度顯著下降（P<0.01），油紅O染色也顯示，雷公藤甲素160 nmol·L^-1組細胞內(nèi)脂滴數(shù)量明顯低于對照組；與對照組比較，雷公藤甲素組TM4細胞內(nèi)TG水平顯著下降（P<0.01）。與雷公藤甲素組比較，使用ATP協(xié)同給藥顯著減輕了雷公藤甲素對TM4細胞存活率的抑制（P<0.01）。與對照組比較，80、160、320 nmol·L^-1的雷公藤甲素作用24 h后，TM4細胞的CD36蛋白表達量顯著上升（P<0.01），160 nmol·L^-1雷公藤甲素作用于TM4細胞6、12、24、36、48 h，CD36的表達水平隨時間的延長先升高后降低，在24 h達到峰值（P<0.05）。與對照組比較，雷公藤甲素組TM4細胞中PKD1及其絲氨酸744-748位點磷酸化水平、HDAC7和FOXO1的蛋白表達均受到顯著抑制（P<0.05、0.01）。結(jié)論 雷公藤甲素對睪丸支持細胞具有明顯的損傷作用，損傷機制與其誘導(dǎo)的脂質(zhì)代謝紊亂和ATP缺乏相關(guān)。CD36在損傷過程中表達量先升高后降低，其初期代償性上升可能是一種細胞的應(yīng)激性保護機制，PKD1/HDAC7/FOXO1信號通路的抑制介導(dǎo)了CD36表達的調(diào)控。

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 BODIPYstaining 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.

R285.5

國家自然科學(xué)基金資助項目（81773992）