艳山姜挥发油自乳化释药系统的毒性及对Caco-2细胞单层细胞旁转运与P-糖蛋白的影响

Toxicity and effects of essential oil of Fructus Alpinia zerumbet-self-emulsifyingdrug delivery system on paracellular transport in Caco-2 cell monolayer and P-glycoprotein

目的 考察艳山姜挥发油自乳化释药系统(essential oil of Fructus Alpinia zerumbet-self-emulsifying drug delivery system,EOFAZ-SEDDS)的毒性及其对人结直肠腺癌Caco-2细胞单层通透性和P-糖蛋白(P-glycoprotein,P-gp)的影响。方法 采用MTT法和乳酸脱氢酶(lactate dehydrogenase,LDH)法测定EOFAZ-SEDDS的细胞毒性;荧光黄渗透率实验考察EOFAZ-SEDDS对Caco-2单层通透性的影响;Western blotting考察EOFAZ-SEDDS对P-gp蛋白表达的影响;流式细胞仪检测EOFAZ-SEDDS对P-gp功能的影响。小鼠ig不同剂量EOFAZ-SEDDS,连续观察14 d,考察EOFAZ-SEDDS对小鼠的急性毒性作用。结果 与EOFAZ组比较,EOFAZ-SEDDS处理培养1、3、5 d的Caco-2细胞24 h后细胞存活率均显著升高(P<0.05、0.01),LDH外漏率显著降低(P<0.05、0.01)。与对照组比较,EOFAZ-SEDDS、单独的EOFAZ均能够显著增加荧光黄透过率(P<0.01);EOFAZ处理Caco-2细胞24 h后,P-gp蛋白表达显著降低(P<0.05),EOFAZ-SEDDS处理后P-gp蛋白表达水平显著升高(P<0.05、0.01);10、20μg/mL的EOFAZ-SEDDS对P-gp外排功能没有影响,40μg/mL EOFAZSEDDS显著增加细胞内罗丹明的荧光强度(P<0.05)。小鼠口服制剂的半数致死剂量(median lethal dose,LD_(50))为5.291 5g/kg,95%置信限为4.927 8~5.682 1 g/kg。根据急性毒性分级标准,EOFAZ-SEDDS对小鼠实际无毒。结论 EOFAZ-SEDDS可以降低EOFAZ的细胞毒性,对小鼠实际无毒,但改变了Caco-2细胞单层屏障功能,同时诱导P-gp表达,在一定浓度范围内对其功能没有影响,为EOFAZ新剂型的研究提供了一定的实验基础。

Objective To evaluate the toxicity and effects of essential oil of Fructus Alpinia zerumbet-self-emulsifying drug delivery system(EOFAZ-SEDDS)on Caco-2 cell monolayer permeability and P-glycoprotein(P-gp).Methods Cytotoxicity of EOFAZ-SEDDS on Caco-2 cell monolayers were detected by MTT and lactate dehydrogenase(LDH)methods.The effect of EOFAZ-SEDDS on Caco-2 cell monolayer permeability was investigated by fluorescence yellow permeability experiment.Western blotting was used to investigate the effect of EOFAZ-SEDDS on P-gp protein expression;The effect of EOFAZ-SEDDS on P-gp function was detected by flow cytometry.Mice were ig different doses of EOFAZ-SEDDS for 14 d,and the acute toxicity of EOFAZ-SEDDS on mice was investigated.Results Compared with EOFAZ group,survival rate of Caco-2 cells cultured for 1,3,5 d after EOFAZ-SEDDS treatment were significantly increased(P<0.05,0.01),and LDH leakage rate was significantly decreased(P<0.05,0.01).Compared with control group,EOFAZ-SEDDS and EOFAZ alone significantly increased the fluorescence yellow transmittance(P<0.01).After EOFAZ treated Caco-2 cells for 24 h,P-gp protein expression was significantly decreased(P<0.05);After EOFAZ-SEDDS treatment,P-gp protein expression was significantly increased(P<0.05,0.01).10 and 20μg/mL of EOFAZ-SEDDS had no effect on the efflux function of P-gp,while 40μg/mL of EOFAZ-SEDDS significantly increased the fluorescence intensity of rhodamine in cells(P<0.05).The median lethal dose(LD_(50))of oral preparations in mice was 5.2915 g/kg,and the 95%confidence limit was 4.9278—5.6821 g/kg.According to the acute toxicity grading standard,EOFAZ-SEDDS was actually non-toxic to mice.Conclusion EOFAZ-SEDDS can reduce the cytotoxicity of EOFAZ,which is actually non-toxic to mice,but it changes the monolayer barrier function of Caco-2 cells and induces the expression of P-gp,which has no effect on its function in a certain concentration range,providing a certain experimental basis for the study of new dosage forms of EOFAZ.