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目的:观察不同浓度复方青娥丸含药血清对去势鼠破骨细胞MMP3-OPN-P53通路蛋白表达及其凋亡的影响,探讨青娥丸抑制骨丢失的作用机制。方法:将20只SD大鼠去势饲养12周,处死选取股骨骨髓,进行体外OC培养。另用含2 g青娥丸生药的药液喂养20只SD大鼠,随机均分为空白对照(A)组(给纯化水1 mL),低浓度(B)组(给药0.5 mL),中浓度(C)组(给药1 mL),高浓度(D)组(给药2 mL),提取各组含药血清干预OC,运用图像系统测定TRAP阳性染色数目和骨磨片陷窝面积,流式细胞仪测定OC凋亡水平,RT-PCR法测定OC通路蛋白MMP3、OPN和P53的mRNA表达水平。结果:(1)术后12周模型组大鼠腰椎及股骨骨密度为(0.159±0.011)g·cm~(-2)和(0.158±0.011)g·cm~(-2),假手术组为(0.330±0.012)g·cm~(-2)和(0.298±0.016)g·cm~(-2),2组有明显差异(P<0.05),表明大鼠骨质疏松的模型建立成功。(2)加入不同药物干预后,D组中MMP3含量明显低于其余各组(P<0.01),OPN和P53含量高于其余各组(P<0.05)。B、C、D是随着含药血清依次增加的3组,MMP3表达依次减少,D组最少,而OPN和P53表达依次增加,其中D组在含药血清药物添加后明显增加,明显不同于A组(P<0.01)。(3)空白对照组流式细胞仪检测无亚G1峰,G0~G1期细胞占23%。低浓度组、中浓度组和高浓度组流式细胞仪均检测在G1期前出一陡立的凋亡峰(亚G1峰),占细胞总数的38%,42%和58%。结论:研究发现青娥丸含药血清可以通过MMP3-OPN-P53信号通路激活OC凋亡,抑制骨丢失,改善骨代谢,防治绝经后骨质疏松症。
OBJECTIVE: To observe the effects of different concentrations of Qing-E-Pill serum on MMP3-OPN-P53 pathway protein expression and apoptosis of osteoclasts in ovariectomized rats, and to explore the mechanism of Qing’e pills inhibiting bone loss. Methods: Twenty SD rats were castrated for 12 weeks. The femoral bone marrows were sacrificed and cultured in vitro. In addition, 20 SD rats were fed with liquid containing 2 g of Qingyang Pill crude drug and randomly divided into control group (A), purified water (1 mL), low concentration (B group) (0.5 mL) (C group) (1 mL) and high concentration group (D group) (2 mL). The serum levels of OC in each group were extracted and the number of TRAP positive staining and the lacunae area The levels of OC apoptosis were measured by flow cytometry. The mRNA expression levels of OC3, OPN and P53 in OC pathway were determined by RT-PCR. Results: (1) The BMD of lumbar vertebral and femur in model group was (0.159 ± 0.011) g · cm ~ (-2) and (0.158 ± 0.011) g · cm ~ (-2) at 12 weeks after operation, (0.330 ± 0.012) g · cm ~ (-2) and (0.298 ± 0.016) g · cm ~ (-2), there was a significant difference between the two groups (P <0.05), indicating that the rat model of osteoporosis was established successfully . (2) After adding different drugs, the content of MMP3 in group D was significantly lower than that in other groups (P <0.01). The content of OPN and P53 in group D was higher than that in other groups (P <0.05). B, C, and D were the three groups which increased in sequence with the drug-containing serum, the expression of MMP3 was decreased in turn, the group D was the least, the expression of OPN and P53 were increased in turn, and the group D was obviously increased after the drug-containing serum was added Group A (P <0.01). (3) There was no sub-G1 peak detected by flow cytometry in blank control group, and 23% of G0 ~ G1 phase cells. In the low concentration group, middle concentration group and high concentration group flow cytometry, a sharp apoptosis peak (sub G1 peak) was observed in G1 phase, accounting for 38%, 42% and 58% of the total number of cells. Conclusion: The study found that Qing-e-pill serum can activate OC apoptosis through MMP3-OPN-P53 signaling pathway, inhibit bone loss, improve bone metabolism and prevent postmenopausal osteoporosis.