补肾通络方对骨质疏松斑马鱼效应评价及破骨细胞自噬机制

Effect of Bushen Tongluo Formula on Osteoporosis of Zebrafish and Study on Autophagy Mechanism of Osteoclast

目的:评价补肾通络方对斑马鱼骨质疏松模型的干预效应,并明确其对破骨细胞分化的自噬调控机制。方法:选取新生3 d健康斑马鱼幼鱼260尾,25 mmol·L-1泼尼松龙(Pred)干预3 d,通过钙黄绿素染色确认建模成功,分为空白组,Pred组(25 mmol·L-1),依替磷酸二钠组(300 mg·L-1),补肾组(180 mg·L-1),通络组(30 mg·L-1)和补肾通络组(210 mg·L-1)各40尾。药物干预4 d后采用钙黄绿素染色后拍照,统计斑马鱼脊椎骨荧光面积;实时荧光定量PCR(Real-time PCR)检测斑马鱼碱性磷酸酶(ALP),骨形态发生蛋白-2b(BMP-2b),Runt相关转录因子2(Runx2),组织蛋白酶K(CTSK),抗酒石酸酸性磷酸酶(TRAP)及活化T细胞核因子(NFATC-1)mRNA表达。培养RAW264.7细胞至80%~90%密度后分为空白组,Rankl诱导组(10μg·L-1),补肾组(180 mg·L-1),通络组(30 mg·L-1)和补肾通络组(210 mg·L-1)。经Rankl诱导细胞破骨分化及药物干预4 d后,采用FITC标记鬼笔环肽染色检测肌动蛋白环表达,Real-time PCR检测RAW264.7细胞TRAP,CTSK和自噬相关基因5(ATG5),自噬相关基因7(ATG7),泛素结合蛋白62(p62)mRNA表达。结果:在体内研究中,与空白组比较,Pred组斑马鱼椎骨面积及ALP,BMP-2b,Runx2表达显著下降(P<0.01),CTSK,TRAP,NFATC-1表达显著升高(P<0.01)。与Pred组比较,通络组椎骨面积明显增加(P<0.05),而补肾通络组椎骨面积显著增加(P<0.01),补肾组,通络组及补肾通络组ALP,BMP-2b,Runx2表达均显著升高(P<0.01),补肾通络组CTSK,TRAP,NFATC-1表达明显降低(P<0.05,P<0.01)。体外研究显示,与空白组比较,Rankl诱导组肌动蛋白环数目显著增多(P<0.01),CTSK,TRAP和ATG5表达显著升高(P<0.01),ATG7,p62表达明显升高(P<0.05)。与Rankl诱导组比较,补肾组、通络组及补肾通络组均明显减少了肌动蛋白环数目及CTSK,TRAP表达(P<0.05,P<0.01),而补肾通络组同时降低了ATG5,ATG7,p62表达(P<0.05,P<0.01)。结论:补肾通络方能够显著改善泼尼松龙诱导的斑马鱼骨质疏松症,并通过降低自噬相关基因表达抑制破骨细胞分化。

Objective:To evaluate the intervention effect of Bushen Tongluo formula on zebrafish osteoporosis model and to clarify its regulation of autophagy mechanism on osteoclast differentiation.Method:The260 young zebrafishes(3 dpf)were selected,the zebrafish osteoporosis model was established with 25 mmol·L-1prednisolone(Pred)for 3 days,confirming the successful model by calcein staining.The zebrafishes were divided into control group,Pred group(25 mmol·L-1),etidronate disodium(ED)group(300 mg·L-1),Bushen(BS)group(180 mg·L-1),Tongluo(TL)group(30 mg·L-1),and Bushen Tongluo(BSTL)group(210 mg·L-1),40 tails per group.After intervened with medicine for 4 days,the calcein staining was adopted to count the vertebral bone fluorescence area of zebrafish,Real-time PCR was adopted to detect the mRNA expression of akaline phosphatase(ALP),bone morphogenetic protein 2b(BMP-2b),runt-related protein 2(Runx2)and cathepsin K(CTSK),phosphorus and tartrate-resistant acid phosphatase(TRAP),nuclear factor of activated T-cells,cytoplasmic-1(NFATC-1).Divided RAW264.7 cells into control,Rankl induction(10μg·L-1),BS(180 mg·L-1),TL(30 mg·L-1),and BSTL group(210 mg·L-1)after they were cultured to 80%-90%density.The expression of actin ring was detected by phalloidin cytoskeleton staining.The mRNA expression of TRAP,CTSK and autophagy-related genes 5(ATG5),autophagy-related genes 7(ATG7),and ubiquitin-binding protein p62(p62)were detected by Real-time PCR.Result:Compared with the control group in vivo,the vertebral area and ALP,BMP-2b,and Runx2 expression of zebrafish in the Pred group were significantly decreased(P<0.01),and CTSK,TRAP,and NFATC-1 expression of zebrafish were significantly increased(P<0.01).Compared with the Pred group,the vertebral area of the TL(P<0.05)and BSTL group(P<0.01)increased significantly.The expressions of ALP,BMP-2b,and Runx2 in the BS,TL,and BSTL group were significant increased(P<0.01).The expression of CTSK,TRAP,and NFATC-1 in BSTL group were significant decreased(P<0.05,P<0.01).Compared with the control group in vitro,the number of actin rings(P<0.01)and CTSK,TRAP,ATG5(P<0.01)expression and ATG7,p62(P<0.01)expression were significantly increased in the Rankl-induced group.Compared with the Rankl induction group,the number of actin rings and CTSK and TRAP expression were significantly decreased in the BS,TL,and BSTL group(P<0.05,P<0.01),while the expression of ATG5,ATG7,and p62 were significant decreased in the BSTL group(P<0.05).Conclusion:BSTL can significantly improve prednisolone-induced zebrafish osteoporosis and inhibit osteoclast differentiation by reducing autophagy-related gene expression.