千金子制霜前后提取物对人胚肾细胞HEK293的体外毒性作用

In vitro Toxicity of Euphorbiae Semen Before and After Crystallization on Human Embryonic Kidney 293 Cells

目的:比较千金子制霜前后提取物对正常人胚肾细胞HEK293的影响,探究千金子制霜减毒的作用机制。方法:采用HEK293细胞为体外模型,随机分为对照组,千金子生品低、中、高质量浓度(100、200、400μg·mL^(-1))组和千金子霜品低、中、高质量浓度(100、200、400μg·mL^(-1))组。细胞体外培养后,分别给予等体积的无血清培养液和千金子生品、霜品提取物溶液,各组均设置3个复孔。采用CCK-8法评价细胞存活率,利用倒置显微镜观察细胞数量及形态变化;碘化丙啶(PI)染色法和AnnexinV-FITC/PI双重染色法检测细胞周期和凋亡情况;试剂盒法检测细胞上清液中乳酸脱氢酶(LDH)、谷胱甘肽(GSH)、肌酐(Cr)和细胞裂解液中尿素氮(BUN)水平。结果:与对照组比较,千金子生品各质量浓度组HEK293细胞存活率降低,细胞形态异常,细胞数量减少,G_(0)/G_(1) 、 G_(1)/G_(2)期细胞比例明显降低,S期细胞比例明显增加,细胞凋亡率升高(P<0.01),LDH、BUN、Cr水平升高(P<0.05,P<0.01),GSH水平降低(P<0.01);与千金子生品组比较,千金子霜品相应质量浓度可显著增加HEK293细胞存活率(P<0.01),改善细胞形态,增加G_(0)/G_(1) 期、 G_(1)/G_(2)期细胞比例,降低S期细胞比例,同时能明显降低LDH、BUN、Cr水平(P<0.05,P<0.01),提高GSH水平(P<0.01)。结论:千金子制霜后可能通过减轻细胞周期阻滞和细胞氧化损伤改善肾细胞功能、减少细胞凋亡,从而降低体外肾毒性。

Objective:To explore the mechanism of Euphorbiae Semen(ES)after crystallization on attenuating toxicity by comparing the effect of ES on human embryonic kidney 293(HEK293)cells before and after crystallization.Methods:HEK293 cells were cultivated as the in vitro model,and they were randomly divided into a control group,three low,medium,and high-dose ES groups(100,200,and 400μg·mL^(-1)),and three low,medium,and high-dose ES Pulveratum(ESP)groups(100,200,and 400μg·mL^(-1)).The cells after cultivation in each group were given equal volumes of serum-free medium,ES extracts,and ESP extracts,respectively,and each group was set up with three duplicate wells.The cell counting kit-8(CCK-8)method was used to evaluate the survival rate of cells,and the inverted microscope was used to observe the number and morphological changes of cells.The propidium iodide(PI)staining and Annexin V-FITC/PI double staining were used to detect the cell cycle and apoptosis.The reagent kit method was used to detect the content of lactate dehydrogenase(LDH),glutathione(GSH),and creatinine(Cr)in cell supernatant,and blood urea nitrogen(BUN)in cell lysate.Results:Compared with the control group,the survival rate of HEK293 cells reduced,and the number of cells decreased with abnormal cell morphology in all ES groups.The ratio of cells in G_(0)/G_(1) and G_(1)/G_(2) phase significantly reduced,whereas that in S phase significantly increased as well as the cell apoptosis rate(P<0.01).In the ES groups,the levels of LDH,BUN,and Cr in SE groups were increased(P<0.05,P<0.01),and the content of GSH was decreased(P<0.01).Compared with the ES group,the corresponding concentration in the ESP groups significantly increased the survival rate of HEK293 cells(P<0.01)and improved cell morphology,thus increasing the ratio of cells in G_(0)/G_(1) and G_(1)/G_(2) phase and decreasing that in S phase.At the same time,it can significantly reduce the levels of LDH,BUN,and Cr were reduced(P<0.05,P<0.01),and activity of GSH was increased(P<0.01).Conclusion:The possible mechanism of ES after crystallization on attenuating nephrotoxicity in vitro is improving renal cell function and reducing cell apoptosis by alleviating cell cycle arrest and cell oxidative damage.