雷公藤甲素-羧化壳聚糖偶联物的体外细胞毒性及其抗炎活性

Evaluation of in vitro cytotoxicity and anti-inflammatory activity of a novel triptolide-carboxylchitosan conjugate

目的:制备并测试一种新型雷公藤甲素-羧化壳聚糖偶联药物(TP-CChit)的体外细胞毒性及抗炎活性。方法:制备并测试TP-CChit的水溶性、体外稳定性和载药量。细胞增殖与毒性试验比较TP和TP-CChit对3种不同细胞活力的影响,流式细胞术检测药物对RAW264.7细胞凋亡的影响。建立脂多糖刺激RAW264.7细胞的炎症模型,通过实时荧光定量PCR(RT-qPCR)检测环氧合酶COX-1,COX-2 mRNA水平的变化。结果:新型TP-CChit药物有较好水溶性、体外稳定性和合适的载药量。TP与TP-CChit对细胞增殖毒性及细胞凋亡的影响与药物浓度及时间成正相关,2种药物对细胞活力影响差异极显著(P<0.001);TP-CChit与TP组相比细胞凋亡率明显下降。LPS炎症模型组,LPS+TP-CChit双处理组分别与对照组COX-1 mRNA水平比较,表达均无显著差异(P>0.05);LPS+TP-CChit双处理组与LPS炎症模型组比较可显著降低COX-2 mRNA的表达水平(P<0.001)。结论:该新型雷公藤甲素-羧化壳聚糖偶联药物大大增强了雷公藤甲素的水溶性、降低了细胞毒性和凋亡水平,对LPS诱导的RAW264.7细胞体外炎症反应具有较好抑制作用。

Objective:To prepare and evaluate a triptolide-carboxylchitosan conjugated drug(TP-CChit)with reduced toxicity and anti-inflammatory activity.Methods:TP-CChit was prepared and the water solubility,in vitro stability and drug loading was determined.TP and TP-CChit were compared for effects on viability of three different kinds of cells by cell proliferation and toxicity assay,and the apoptosis of RAW264.7 cells was detected by flow cytometry.An inflammatory model of lipopolysaccharide-stimulated RAW264.7 cells was established,and the mRNA levels of inflammatory factors COX-1 and COX-2 were detected by RT-qPCR.Results:TP-CChit had good solubility,stability and suitable drug loading.The effects of TP and TP-CChit on cell viability and apoptosis were positively correlated with drug concentration and stimulation time.The effects of the two drugs on cell viability were extremely different(P<0.001).The apoptosis rate of TP-CChit was significantly reduced.LPS+TP-CChit double treatment group had no significant difference in COX-1 mRNA level compared with the control group(P>0.05).LPS+TP-CChit double treatment group significantly reduced the expression level of COX-2 mRNA compared with the LPS inflammation model group(P<0.001).Conclusion:TP-CChit greatly enhances the water solubility of triptolide,reduces the cytotoxicity and apoptosis level,and has a good inhibitory effect on LPS-induced RAW264.7 cell inflammatory response.