摘要
目的:观察中药复方保肝宁对瘦素刺激的肝星状细胞(Hepatic stellate cells,HSCs)增殖功能的影响及其发生的可能机制。方法:实验于2005-07/2006-07在南方医科大学细胞生物实验室完成。①药物血清的制备:保肝宁的方药由黄芪、桃仁、丹参、黄芩、白背叶根、鳖甲、柴胡、白芍、枳实等组成,煎煮配制成1.77g/mL,3.54g/mL剂量药液。秋水仙碱配制成0.05g/mL。取wistar大鼠24只,随机分为4组,每组6只,分别为正常血清组、保肝宁等剂量组、保肝宁二倍剂量组、秋水仙碱含药血清组。正常血清组给予等量生理盐水,其余各组给予相应药物1mL/100g,胃灌1次/d,连续7d。腹主动脉采血,离心分离血清。②用四甲基偶氮唑盐比色法检测0,10,50,100,200及400μg/L的瘦素在6,12,18,24h对HSC-LX2增殖的影响及保肝宁对100μg/L的瘦素刺激HSC-LX2增殖的阻断作用;应用蛋白印迹实验检测细胞膜瘦素受体长片段b(OB-Rb)蛋白表达水平。结果:①不同剂量瘦素在不同时间点对HSC-LX2增殖的影响:瘦素剂量为10～400μg/L时,对HSC-LX2的促进增殖作用呈现剂量、时间依赖性。各剂量组之间差异有显著性(P<0.01),同一剂量、不同时间比较差异有显著性(P<0.01)。②保肝宁对HSC-LX2生长的影响:与正常血清组比较,保肝宁等剂量组、保肝宁二倍剂量组均能明显抑制HSC-LX2的增殖(A600值,0.818±0.239,0.115±0.114,0.107±0.055,n=5,P<0.01),秋水仙碱组(0.388±0.073)亦能抑制HSC-LX2细胞的增殖(P<0.05);与秋水仙碱组比较,保肝宁等剂量组、保肝宁二倍剂量组明显抑制HSC-LX2的增殖(P<0.05)。③保肝宁作用HSC-LX20不同时间后OB-Rb的表达:与正常血清组,保肝宁作用6h后,OB-Rb水平有显著的降低,秋水仙碱组则无明显的降低。结论:中药复方保肝宁有阻断瘦素刺激HSCs-LX2增殖的作用,而OB-Rb的表达降低可能是其主要的作用机制之一。
AIM： To investigate the effect of Baoganning （BGN） on the proliferation of hepatic stellate cells （HSCs） stimulated by leptin and its relevant mechanisms. METHODS： The experiment was conducted at the Laboratory of Ceils, Southern Medical University between July 2005 and July 2006. （1） Preparation for drug serum： BGN was composed of Milkvetch Root, Peach Seed, Danshen root, Baical Skullcap Root, Whitebackleaf Mallotus Root, Turtle Shell, RadixBupleuri, White Peony Root, and Immature Bitter Orange. It was decocted and confected to 1.77 g/mL, 3.54 g/mL liquid. Colchicine was confected to 0.05 g/mL liquid. Twenty-four Wistar rats were randomly assigned into 4 groups with 6 rats in each group： Normal serum group, BGN dosage group, BGN double dosages group, and Colchicine group. Rats in the normal serum group were administrated with normal saline at the same dosage, while rats in the other groups were gastdcally perfused with 1 mL/100 g corresponding drugs once a day for 7 continuous days. The blood was obtained from the abdominal aorta to separate the serum. （2） The effects of leptin of 0, 10, 50, 100, 200 and 400 μg/L on the proliferation of HSC-LX2 at the 6^th, 12^th, 18^th, and 24^th hours as well as the effect of BGN in blocking the proliferation of HSC-LX2 stimulated by leptin were measured by MTT assay. The protein expression of OB-Rb was detected by Western blot method. RESULTS： （1） Effects of leptin at different doses and different time points on the proliferation of HSC-LX2： The promoting effects of leptin at the doses of 10-400μg/L were in dose-dependent and time-dependent manners, and there were distinguished differences among all dose groups （P 〈 0.01）. Similarly, there were distinguished differences in the same group among different time points （P 〈 0.01）. （2） The effect of BGN on the multiplication of HSC-LX2： Compared with normal serum group, the multiplication of HSC-LX2 in BGN dose group and BGN double doses group were significantly restrained （on A600, 0.818±0.239,0.115±0.114,0.107±0.055,n =5, P 〈 0.01）. Colchicine group（0.388±0.073）could also restrain the multiplication of HSC-LX2 （P 〈 0.05）. Compared to the Colchicine group, both of the BGN groups could remarkably restrain the multiplication of HSC-LX2 （P 〈 0.05）. （3） The expression of OB-Rb at different time points in the BGN groups： Compared with the normal serum group, the expression of OB-Rb at 6 hours after BGN＇s effect was greatly decreased, but not in the Colchicine group. CONCLUSION： BGN can inhibit the proliferation of HSCs-LX2 stimulated by leptin, and decreased expression of OB-Rb might he one of the mechanisms.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第7期1290-1292,共3页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家自然基金资助课题(30672769)
广东省中医药局基金资助课题(1050128)~~
