基于靶标循环放大策略的双色荧光传感体系探索何首乌“九蒸九晒”炮制过程中不同蒸制次数对肾细胞的减毒作用

A dual-color fluorescence sensing system based on target cycle amplification strategy to explore attenuating effect of different steaming times on kidney cells in process of“nine times steaming and nine times sunning”of Polygoni Multiflori Radix

目的通过采用所建立的双色荧光传感体系nanoflare测定肾细胞分泌的miR-21和miR-200c表达,观察经“九蒸九晒”炮制后的制首乌对肾细胞的毒性作用。方法生首乌和不同炮制次数制首乌的水提物与肾细胞孵育后,利用双色荧光传感体系nanoflare对给药前后细胞培养基中的miR-21和miR-200c进行定量检测。结果通过比较不同炮制次数的何首乌水提物的毒性,发现伴随着炮制次数的增加,双色荧光传感体系的荧光恢复强度在不断减弱,制首乌水提物处理后的细胞培养基中miR-21和miR-200c的表达在不断减少,说明在炮制过程中毒性逐渐减弱,炮制至第9次时毒性明显减弱,对肾细胞未产生明显毒性,表明制首乌水提物对肾细胞的毒性随炮制次数增加在不断降低。结论生首乌毒性很强,而经炮制后的制首乌毒性逐渐减弱,在炮制次数超过9次后基本无毒,表明通过“九蒸九晒”在细胞水平可以达到减毒的作用,为制首乌炮制工艺与质量标准的完善提供参考。

Objective To detect the expressions of miR-21 and miR-200c secreted by kidney cells by using the established dual-color fluorescence sensing system,and observe the effects of decrease toxicity of processed Heshouwu(Polygoni Multiflori Radix)after“nine times steaming and nine times sunning”on kidney cells.Methods After kidney cells being incubated with water extracts of crude and processed Polygoni Multiflori Radix with different preparation times,two-color fluorescence sensor system was used to quantitatively detect miR-21 and miR-200c in cell culture media before and after administration.Results By comparing the toxicity of the water extracts of Polygoni Multiflori Radix with different processing times,with the increasing of processing times,the fluorescence recovery intensity of two-color fluorescence sensor system was constantly weakened,and expressions of miR-21 and miR-200c in cell culture media treated with water extracts of processed Polygoni Multiflori Radix was reduced,which indicating that toxicity gradually weakened during the processing.The toxicity was significantly weakened at the ninth time,and the toxic effect on kidney cells was not significant,indicating that the toxicity of water extracts of processed Polygoni Multiflori Radix to kidney cells was continuously reduced with the increasing of processing times.Conclusion Raw Polygoni Multiflori Radix is toxical,but the toxic of processed Polygoni Multiflori Radix is gradually weakened.After processing more than nine times,processed Polygoni Multiflori Radix is basically non-toxic,indicating that“nine times steaming and nine times sunning”can achieve attenuation at cell level,which can provide a reference for the improvement of processing technology and quality standards of Polygoni Multiflori Radix.