葛根素预防酒精性股骨头坏死的实验研究

Experimental study to prevent alcohol-induced osteonecrosis of the femoral head with puerarin

目的:从细胞学和动物学实验观察葛根素预防酒精性股骨头坏死的作用。方法:①细胞学实验:分离培养小鼠骨髓基质细胞,随机分为3组:A.模型组(给予酒精0.09mol/L处理细胞),B.实验组(酒精0.09mol/L和葛根素终浓度为0.01mg/ml),C.对照组(无酒精和葛根素)。苏丹Ⅲ染色,光镜下脂肪细胞计数;测定细胞内甘油三酯含量、碱性磷酸酶活性和细胞培养液中的骨钙素含量。10天后收集细胞,采用逆转录聚合酶链式反应(RT-PCR)技术,检测各组细胞中成脂转录因子PPARγ mRNA和成骨基因Osteocalcin mRNA的表达。②动物学实验:取4周龄健康昆明小白鼠300只,随机分为3组,A组:灌胃法给予烈性白酒(含乙醇46%)20mL/(kg.d),肌注Ns10ml/(kg.d);B组:同法给予等量白酒,肌注葛根素注射液0.5g/(kg.d);C组(对照组):同法给予普通饮用水20mL/(kg.d),肌注NS10ml/(kg.d)。于实验4、6、8、10个月分批处死动物,检测动物的血清学、肝脏和股骨头组织学、基因表达等变化。结果:(1)细胞学实验:上述处理细胞并培养21天后,实验组中脂肪细胞数量、细胞内甘油三酯含量均明显低于模型组(P<0.001),且与对照组差异不显著(P>0.05)。上述处理细胞并培养12天后实验组中细胞内碱性磷酸酶活性、细胞培养14天后培养液中骨钙素含量,均明显高于模型组(P<0.001),且与对照组差异不显著(P>0.05)。实验组和对照组细胞中PPARγmRNA的表达明显低于模型组(P<0.05),而对照组与实验组间的差异无显著性(P>0.05)。实验组和对照组细胞中OsteocalcinmRNA表达明显增高,分别是模型组的1.9倍和2.4倍(P<0.01),而实验组与对照组间的差异无显著性(P>0.05)。(2)动物学实验:实验6个月时:①3组血清CHO、TG、ALP分别为:(6.39±0.49)、(3.19±0.11)、(2.98±0.36)mmoL/L,(1.01±0.15)、(0.71±0.13)、(0.68±0.22)mmol/L,(161.6±32.44)、(196.5±31.52)、(203.4±22.83)IU。②A组出现脂肪肝,股骨头骨小梁变细稀疏,髓内造血组织减少,脂肪与空骨陷窝明显增多。B组股骨头内脂肪稍增加,空骨陷窝比正常C组少。3组最大脂肪细胞平均直径和空骨陷窝计数分别为(40.02±3.25)、(39.15±3.67)、(38.51±3.09)μm,(13.5±1.6)、(9.8±2.2)、(10.3±2.7)%。③A组中PPARγmRNA呈高表达,Osteocalcin mRNA呈低表达。B组和C组中PPARγ mRNA呈低表达,Osteocalcin mRNA呈高表达。始自6个月,A组各数据和B、C组间差异均有统计学意义(P<0.05,P<0.01),B、C组差异无统计学意义(P>0.05)。结论:葛根素能够对抗酒精的毒性作用,抑制酒精诱导骨髓基质细胞的成脂分化,维持其成骨分化,能够预防小鼠酒精性ONFH的发生。

Objective： To prevent alcohol-induced osteonecrosis of the femoral head, observe the effects of inhibition of puerarin on differentiation of marrow stromal cells into adipocytes induced by alcohol by the methods of cell biology and animal experiment. Methods ： ① Cell biology ： Marrow stromal cells （MSCs） were isolated from mice bone marrow, obtained and cultured. MSCs were separated by 3 groups at random ： Group A ： the cells were treated with 0.09moL/L alcohol. Group B：the cells were treated with 0.09moL/L alcohol and 0.01mg/ml puerarin. Group C：the cells were treated without alcohol or puerarin as control. The cells in culture were stained with Sudan Ⅲ, and then the number of adipocytes were counted on a light microscope. The contents of triglyceride and alkaline phosphatase activity in cells were determined by biochemical assay; The contents of osteocalcin in the media were determined by radioimmunoassay. Cells were collected after 10 days,the expression levels of the peroxisome proliferator-actived receptor-γ （PPARγ） mRNA, and osteocalcin mRNA in cells were analyzed by Reverse Transcription - Polymerase Chain Reaction （RT-PCR）. ②Animal experiment：300 of 4 week-old mice were divided into 3 groups by random. In group A, the distilled spirit containing 46% of alcohol was administered intragastrically at a dose of 20ml/（ kg · d） ,and the normal saline at dosage of 10ml/（kg · d） by intramuscular injection. In group B, the same dose of the distilled spirit was administered intragastrically, and puerarin at dosage of 0.5g/（kg · d） by intramuscular injection. In group C,the water was administered intragastrically at a dose of 20mL/（ kg · d） ,and the same dose of normal saline were administered as control by same method. The animals were killed in batches in the 4,6,8,10 months. The changes of serology, livers and bilateral femoral heads, adipogenesis and osteogenesis gene expression of the cells in femoral head of mice were detected. Results：①Cell biology：In vitro,the number of adipocytes and the contents of triglyceride in the cells treated for 21 days in Group B were significantly lower than that in Group A. The alkaline phosphatase activity values,the cells were treated for 12 days,and the contents of the osteocalcin in the media,the cells were treated for 14 days,in Group B were significantly higher than that in Group A （ P 〈 0. 001 ）. There were not significant differences statistically between Group B and Group C （ P 〉 0.05 ）. The expression levels of PPARγ mRNA in the cells of Group B and Group C were significantly lower than that in the cells of Group A （ P 〈 0.05 ）, and there were not significant differences statistically between Group B and Group C （ P 〉 0.05 ）. The expression levels of osteocalcin mRNA in the cells of Group B and Group C were significantly higher than that in the cells of Group A （ P 〈 0.01 ）, which were 1.9 and 2.4 fold higher than that in Group A respectively, and there were not significant differences statistically between Group B and Group C （ P 〉 0.05 ）. ②Animal experiment： In vivo, When re.ice were treated 6 months： ①The contents of CHO, TG in serum, and ALP in group A, B, C were （6.39 ±0.49） ,（3.19 ±0.11）,（2.98 ±0.36）mmol/L;（1.01 ±0.15） ,（0.71 ±0.13） ,（0.68 ± 0.22 ） mmol/L ; （ 161.6 ± 32.44 ）, （ 196.5 ±31.52 ）, （ 203.4 ± 22.83 ） IU respectively. ②In group A, the thinner and sparse trabeculae, diminished hematopoiesis,fatty tissue and empty osteocyte lacunae increased in the subchondral area of the femoral head, and fatty liver were found. In group B, fatty tissue of the femoral head slightly increased, and near to that in group C that was normal. The mean of the biggest adipocytes diameter and empty osteocyte lacunae in 3 groups was （40.02 ±3.25） ,（39.15 ±3.67） ,（38.51 ± 3.09） μm, （13.5 ± 1.6） ,（9.8 ±2.2） ,（10.3 ± 2.7 ） % respectively. ③There was high expression of PPARγ mRNA and low expression of osteocaosin mRNA in the cells of femoral head in group A. There was low expression of PPART mRNA （ P 〈 0.05） and high expression of osteocaosin mRNA （ P 〈 0.01 ） in group B and group C. From 6 months, there were statistically significant differences compared the results in group A to group B and in group C （P 〈0.01 ） ,and not statistically significant differences between group B and group C （ P 〉 0.05 ）. Conclusion ： Puerarin could against the toxicity of alcohol, inhibit differentiation of marrow stromal cells into adipocytes induced by alcohol, to maintain the normal procedure of osteogenic differentiation, which may prevent the development of alcohol-induced osteonecrosis of the femoral head.