纳米脂质体制剂提高苦参碱去活化肝星状细胞作用研究

Study on nanoliposomal agents in improving effect of matrine on liver fibrosis

目的研究通过制备纳米脂质体提高苦参碱(matrine,MT)去活化肝星状细胞的体外作用效果。方法制备不同载药量(5%、10%、20%)的苦参碱纳米脂质体(Nano-MT)并进行表征;分析药物体外释放行为;给予LX-2细胞不同浓度的Nano-MT(350.00、175.00、87.50、43.75、21.80、10.90、5.40、2.70、1.40 mmol/L),MTT法检测细胞存活率,计算半数抑制浓度(IC50);考察MT、Nano-MT(10 mmol/L)对人肝脏星状细胞LX-2的去活化效果,细胞组化染色检测平滑肌肌动蛋白(α-SMA)、转化生长因子-β(TGF-β)表达;实时荧光定量PCR(qRT-PCR)法检测α-SMA、TGF-β、成纤维细胞生长因子(FGF)、成纤维细胞活化蛋白(FAP)mRNA表达。结果Nano-MT透射电镜表征下呈圆形颗粒状;载药量10%制剂载药量较高,粒径分布均一,稳定性好,满足给药需求;制剂中药物释放符合Higuchi方程(R2=0.9513),72 h左右药物释放量趋于饱和,累计释放量为92.3%。与对照组和MT组比较,Nano-MT组α-SMA、TGF-β阳性细胞数均显著下降(P<0.01、0.001),α-SMA、TGF-β、FGF、FAP mRNA水平显著降低(P<0.01、0.001)。结论Nano-MT具有一定的缓释作用;可以通过抑制α-SMA表达去活化LX-2细胞抑制肝纤维化。

Objective To study the effect of improving matrine(MT)on deactivating hepatic stellate cells and inhibiting liver fibrosis in vitro by nanoliposome delivery technology.Methods Matrine nanoliposomes(nano-MT)with different drug loading(5%,10%,20%)were prepared and characterized.The drug release behavior in vitro was analyzed.LX-2 cells were given different concentrations of nano-MT(350.00,175.00,87.50,43.75,21.80,10.90,5.40,2.70,1.40 mmol/L),the cell survival rate was detected by MTT method,and the half inhibitory concentration(IC50)was calculated.The expression ofα-SMA and transforming growth factor-β(TGF-β)was detected by histochemical staining.The mRNA expression ofα-SMA,TGF-β,fibroblast growth factor(FGF)and fibroblast activating protein(FAP)were detected by real-time quantitative PCR(qRT-PCR).Results Matrine loaded nanoliposome are round particles under transmission electron microscope characterization.The drug loading of 10%preparation was high,the particle size distribution was uniform,and the stability was good,which can meet the needs of drug delivery.Drug release profile of the formulation conforms to the Higuchi equation(R2=0.96513).Drug release tends to 92.3%at time point of 72 h.Compared with control and MT group,the number ofα-SMA and TGF-βpositive cells in nano-MT group were significantly decreased(P<0.01 and 0.001),and the mRNA levels ofα-SMA,TGF-β,FGF and FAP were significantly decreased(P<0.01 and 0.001).Conclusion Martrine loaded liposomes have good pharmaceutical properties and have a certain sustained release effect.The liposome can inhibit liver fibrosis by effectively inhibiting the expression ofα-SMA in liver stellate cells,thereby inhibiting liver fibrosis.