摘要
目的:探讨骨痹通消颗粒对激素性股骨头坏死过程中细胞凋亡的影响,揭示其防治糖皮质激素(glucocorticoids,GCs)诱导骨坏死疾病的机制。方法:将12只新西兰兔随机分为3组,对照组、模型组和实验组,每组4只。除对照组外,其余组使用脂多糖联合糖皮质激素制备股骨头坏死动物模型,造模成功后,实验组给予骨痹通消颗粒灌胃,模型组及对照组给予等量生理盐水,1次/d,持续8周后处死实验动物,取出双侧股骨头,通过HE染色观察病理学改变。体外培养小鼠胚胎成骨细胞MC3T3-E1,使用1×10-5mol/L地塞米松(dexamethasone,Dex)制备细胞模型,实验组在此基础上加入不同体积分数骨痹通消颗粒含药血清诱导培养,对照组常规培养。CCK-8法观察细胞活力,LDH释放实验、TUNEL染色法观察细胞凋亡情况,Western blot和RT-qPCR法分析各组目标蛋白及基因的表达情况。结果:HE染色结果显示,对照组与实验组骨小梁面积显著高于模型组,且股骨头空骨陷窝率显著低于模型组(P<0.01)。体外细胞实验表明,骨痹通消颗粒含药血清可促进MC3T3-E1细胞增殖且对Dex诱导的细胞凋亡具有明显的保护作用(P<0.05)。当含药血清体积分数为5%时可明显减少TUNEL染色后的阳性细胞数。此外,与模型组对比,5%的含药血清能够抑制促凋亡基因Bax的表达,上调抗凋亡基因Bcl-2的表达量(P<0.01),同时提高通路蛋白AKT磷酸化水平,下调凋亡蛋白Caspase-3和Caspase-9的表达(P<0.05)。结论:骨痹通消颗粒能够降低实验动物骨组织中空骨陷窝率,维持骨量,预防股骨头塌陷;此外,通过促进成骨细胞发育,激活PI3K/AKT通路,调控Bax/Bcl-2复合体,下调由GCs介导的Caspase高表达进而减少成骨细胞凋亡,可有效延缓股骨头坏死的进程。
Objective:To investigate the effect of Gubi Tongxiao Granule on apoptosis of osteonecrosis of femoral head induced by glucocorticoids(GCs),and to reveal its mechanism in preventing and treating GCs-induced osteonecrosis.Methods:12 New Zealand rabbits were randomly divided into the control group,the model group and the study group,with 4 rabbits in each group.The model of femoral head necrosis was established by lipopolysaccharide and glucocorticoid.After successful modeling,the study group was treated with Gubi Tongxiao Granule,and the model group and the control group were given the same amount of normal saline,once per day for 8 weeks.HE staining was used to observe the pathological changes.Mice embryonic osteoblasts MC3T3-E1 were cultured in vitro,and Dexamethasone(Dex,10-5 mol/L)was used to prepare the cell model.On this basis,the study group was induced by adding different volume fractions of serum containing Gubi Tongxiao Granule,whereas the control group was cultured routinely.Cell viability was observed by CCK-8 assay,apoptosis was observed by LDH release assay and TUNEL staining,and the expressions of target proteins and genes were analyzed by Western blot and RT-qPCR.Results:HE staining showed that the trabecular bone area was significantly larger,and the empty bone lacunae rate of the femoral head was significantly lower in the control group and the study group than those in the model group(P<0.01).In vitro cell experiments showed that serum containing Gubi Tongxiao Granule could promote the proliferation of MC3T3-E1 cells and had a significant protective effect on Dex-induced cell apoptosis(P<0.05).When the volume fraction of drug-containing serum was 5%,the number of positive cells after TUNEL staining was significantly reduced.In addition,compared with the model group,5%drug-containing serum could inhibit the expression of pro-apoptotic gene Bax,up-regulate the expression of anti-apoptotic gene Bcl-2(P<0.01),increase the phosphorylation level of pathway protein AKT,and down-regulate the apoptotic protein expressions of Caspase-3 and Caspase-9(P<0.05).Conclusion:Gubi Tongxiao Granule can reduce the hollow bone lacunae rate,maintain bone mass,and prevent femoral head collapse in experimental animals.In addition,the process of femoral head necrosis can be effectively delayed by promoting osteoblast development,activating PI3K/AKT pathway,regulating Bax/Bcl-2 complex,down-regulating high expression of Caspase mediated by GCs and reducing osteoblast apoptosis.
作者
朱彩玉
朱磊
顾一帆
刘涛
王志鹏
侯文渊
周正新
ZHU Caiyu;ZHU Lei;GU Yifan;LIU Tao;WANG Zhipeng;HOU Wenyuan;ZHOU Zhengxin(The First Clinical Medical College,Anhui University of Chinese Medicine,Hefei 230031,China;The First Affiliated Hospital of Anhui University of Chinese Medicine,Hefei 230031,China)
出处
《中医药学报》
CAS
2023年第6期31-37,共7页
Acta Chinese Medicine and Pharmacology
基金
安徽省自然科学基金面上项目(2008085MH281)
安徽省教育厅高校自然科学研究重点项目(KJ2020A0403)。
