仙茅苷调控成骨细胞分化对骨质疏松治疗作用的动物实验

Study on the effect of curculigoside on osteoporosis

目的探讨仙茅苷对MG63细胞成骨分化的调节以及对骨质疏松小鼠的保护作用。方法采用甲基噻唑基四唑(methyl thiazolyl tetrazolium,MTT)法检测仙茅苷对地塞米松或H2O2处理的MG63细胞存活率的影响,每种处理均分为6组:空白对照组、空白给药组(5.0μmol/L仙茅苷)、模型组(地塞米松或H2O2处理组)、低剂量给药组(地塞米松或H2O2+1.0μmol/L仙茅苷)、中剂量给药组(地塞米松或H2O2+2.5μmol/L仙茅苷)、高剂量给药组(地塞米松或H2O2+5.0μmol/L仙茅苷),每组样本量为10。蛋白质印迹法检测0、1.0、2.5和5.0μmol/L仙茅苷孵育1、7和14d后MG63细胞成骨分化相关蛋白[Ⅰ型胶原、整合素β1、成骨细胞特异性转录因子(Osterix)、骨钙蛋白和骨桥蛋白]的表达,每组每个时间点样本量为6。将实验小鼠分为4组:空白组、骨质疏松模型组(地塞米松处理)、仙茅苷低剂量组(地塞米松+5mg/kg仙茅苷处理)和高剂量组(地塞米松+45mg/kg仙茅苷处理),每组20只;各组相应处理后对4组小鼠进行胫骨HE染色,计数破骨细胞数量,酶联免疫吸附测定法检测血清中氧化相关因子水平。结果MTT结果显示,与空白对照组[(100±3.7)%]相比,地塞米松制备的模型组细胞存活率减小至(44.1±5.7)%(P<0.05),相应高剂量给药组细胞存活率恢复至(79.7±3.8)%;H2O2处理后的模型组细胞存活率减小至(59.1±4.7)%(P<0.05),相应中剂量给药组细胞存活率恢复至(80.8±3.5)%。蛋白质印迹法结果显示,与0μmol/L仙茅苷处理相比,1.0、2.5和5.0μmol/L仙茅苷孵育1、7和14d后MG63细胞Ⅰ型胶原、整合素β1的表达均显著增加(P<0.05),孵育1d后Osterix和骨钙蛋白的表达均显著增加(P<0.05);而与0μmol/L仙茅苷处理相比,1.0、2.5、5.0μmol/L仙茅苷孵育7和14d后MG63细胞骨桥蛋白的表达差异均无统计学意义(P>0.05)。与空白组相比,骨质疏松模型组小鼠胫骨破骨细胞数量增加;仙茅苷低剂量和高剂量组小鼠胫骨骨皮质更连续且破骨细胞数量减少;与空白组相比,骨质疏松模型组小鼠活性氧显著增加(P<0.05),超氧化物歧化酶、过氧化氢酶显著减小(P<0.05)。与骨质疏松模型组相比,仙茅苷低剂量组和高剂量组小鼠活性氧均显著减小(P<0.05),超氧化物歧化酶、过氧化氢酶均显著增加(P<0.05)。结论仙茅苷通过增加成骨分化相关蛋白表达促进MG63细胞分化,同时对地塞米松诱导的骨质疏松小鼠有一定的治疗作用。

Objective To investigate the regulation of curculigoside on osteogenic differentiation of MG63 and the protective effect on osteoporosis model mice. Methods The effects of curculigoside on the survival rate of dexamethasone or H2O2 treated MG63 were detected by methyl thiazolyl tetrazolium (MTT). The specimens were divided into six groups: blank control group, blank administration group, model group (dexamethasone or H2O2 treatment group), low dose group (dexamethasone or H2O2+1.0 μmol/L curculigoside), medium dose group (dexamethasone or H2O2+2.5 μmol/L curculigoside) and high dose group (dexamethasone or H2O2+5.0 μmol/L curculigoside), the sample size of each group was 10. Western blotting was used to detect the expression of osteogenic differentiation-related proteins [type Ⅰ collagen, integrin β1, osteoblast-specific transcription factor (Osterix), osteocalcin and osteopontin] in MG63 cells after 1, 7 and 14 days incubated with 0, 1.0, 2.5 and 5.0 μmol/L of curculigoside. The sample size for each group at each time point was six. The experimental mice were divided into 4 groups: blank group, model group (dexamethasone treatment group), curculigoside low-dose group (dexamethasone+5 mg/kg curculigoside) and high-dose group (dexamethasone+45 mg/kg curculigoside), twenty each. After treatment, the tibia of the mice in each group were subjected to sacral HE staining. The number of osteoclasts was counted, and the levels of oxidative related factors in serum were determined by enzyme-linked immunosorbent assay (ELISA). Results The MTT results showed that compared with the blank control group [(100±3.7)%], the cell survival rate decreased to (44.1±5.7)% after treatment with dexamethasone, and the survival rate increased to (79.7±3.8)% after treatment with 5.0 μmol/L of curculigoside. The cell survival rate decreased to (59.1±4.7)% after H2O2 treatment, and the survival rate increased to (80.8±3.5)% after treatment with 2.5 μmol/L of curculigoside. The results of Western blotting showed that the expression of type Ⅰ collagen and integrin β1 in MG63 cells was significantly increased after 1.0, 2.5 and 5.0 μmol/L of curculigoside for 1, 7 and 14 days compared with 0 μmol/L of curculigo side for the same period. After increasing (P<0.05), the expression of Osterix and osteocalcin was significantly increased after 1 day of incubation (P<0.05). However, compared with 0 μmol/L curculigoside treatment, the expression of osteopontin in MG63 cells was not significantly different after incubation with 1.0, 2.5, 5.0 μmol/L of curculigoside for 7 and 14 days (P>0.05). Compared with the blank group, the number of tibia osteoclasts in the osteoporosis model group increased. In the low-dose and high-dose groups of curculigoside, the tibia cortex was more continuous and the number of osteoclasts decreased. Compared with the blank group, the activity of oxygen in the osteoporosis model group was significantly increased (P<0.05), and superoxide dimutase and catalase were significantly decreased (P<0.05). Conclusions Curculigoside promotes the differentiation of MG63 cells by increasing the expression of osteoblast differentiation-related proteins, and has a certain therapeutic effect on dexamethasone-induced osteoporosis mice.