麝香配伍乳香促前列腺上皮屏障通透性作用的实验研究

Experimental study on Musk and Olibanum to promote the permeability on prostatic epithelial barrier

目的:应用RWPE-1细胞单层模型体外模拟人体前列腺上皮屏障,分析探讨芳香开窍药物麝香配伍乳香对前列腺上皮屏障的促通透作用。方法:利用人前列腺上皮细胞RWPE-1制备细胞单层模型,以梯度浓度(2.5%,5.0%,10.0%,20.0%)麝香配伍乳香含药血浆(DP-M&O)处理后检测细胞单层电阻TEER值与细胞毒性,采用酚红透过实验观察RWPE-1细胞单层细胞连接和通透性变化,并采用免疫荧光与Western Blot方法检测与细胞紧密连接功能相关的ZO-1蛋白表达情况。结果:经DP-M&O处理后,细胞单层电阻TEER值降低,且呈现明显时效关系,以30-60min时间段及10.0%,20.0%浓度时下降最明显;细胞毒性实验表明,2.5%、5.0%浓度DP-M&O无细胞毒作用,细胞单层的细胞膜可保持完整性不受影响,高浓度10.0%、20.0%DP-M&O可使细胞膜的通透性增加,在24h时变化最为明显,而后进入平缓期;随着DP-M&O浓度增加,基底侧渗透过的酚红含量增加,吸光度OD值明显增高,但10.0%与20.0%两浓度组间差异无统计学意义,表明DP-M&O对酚红透过的影响无剂量依赖性;ZO-1蛋白表达检测结果显示,10.0%、20.0%浓度DP-M&O处理后,RWPE-1细胞ZO-1表达的免疫荧光减少,ZO-1表达量明显下降(P<0.05,P<0.01),但10.0%、20.0%两浓度组间比较差异无统计学意义。结论:麝香、乳香具有促进前列腺RWPE-1细胞单层紧密连接通透性的作用,在高剂量(10.0%-20.0%)含药血浆浓度时作用明显,但无剂量依赖性,24h时增加明显,随时间持续延长,促通透作用可缓慢上升。

Objective： To investigate the the effects of resuscitation-inducing aromatic herbs of Musk and Olibanum to promote permeability on prostatic epithelial barrier with RWPE-1 cell monolayer model. Methods： The RWPE-1 cells were used to prepare as a cell monolayer model which had the function of epithelial tight junction like the human prostatic epithelial barrier in vitro, and treated with increasing dilution （2.5%, 5.0%, 10.0%, 20.0%） of drug plasma of Musk and Olibanum （DP-M＆O）. Thus the value of cell resistance TEER and cytotoxicity were examined. The changes of cell junction and permeability of cell monolayer were analyzed by phenol red test. The ZO-1 protein expression associated with cell junction function was examined by immunofluorescence and Western blot. Results： The TEER of cell monolayer decreased after treatment with DP-M＆O and showed significant time-effect relationship with results of most significant decreasing in time points of 30-60 min and dosages of 10.0% and 20.0%. The cytotoxicity data showed that cell monolayer could keep integrity while treated with 2.5%-5.0% dilution of DP-M＆O, and reversely its permeability increased when treated with 10.0%-20.0% dilution of DP-M＆O. The crossing rates of phenol red through cell monolayer and OD of absorption increased with increasing dilution of DP-M＆O but no difference between dilution 10.0% and 20.0% of it. After treatment with DP-M＆O, the protein expression and immunofluorescence intensity of ZO-1 decreased in dilution of 10.0% and 20.0% （P〈0.05, P〈0.01）, but there was no statistically significance between them. Conclusion： Musk and Olibanum have the role of permeability promotion of cell monolayer fight junction without dosage dependence. Those functions increased slowly with prolong of time and got peak in 24h time point.