基于TGF-β/Smad与MAPK/NF-κB信号通路探讨龟鹿二仙胶对去势大鼠骨质疏松的保护作用

Explore on Protective Effect of Guilu Erxian Jiao on Osteoporosis in Ovariectomized Rats Based on TGF-β/Smad and MAPK/NF-κB Signaling Pathway

关键词:  龟鹿二仙胶  骨质疏松症  TGF-β/Smad  MAPK/NF-κB

Key words:  Guilu Erxian Jiao  osteoporosis  TGF-β/Smad  MAPK/NF-κB

摘要:

目的 评价龟鹿二仙胶对去势大鼠骨质疏松的保护作用并探讨其机制。方法 120只大鼠，随机分为正常组，模型组，龟鹿二仙胶低、中、高剂量组和阿法骨化醇组，共6组，除正常组外均去势造模。龟鹿二仙胶按低、中、高剂量（0.5，1.5，2.5 g·kg^-1·d^-1），阿法骨化醇按0.1 μg·kg^-1·d^-1分别灌胃干预，正常组和模型组分别给予等量生理盐水。采用HE染色检测大鼠股骨病理改变，采用试剂盒检测股骨钙（calcium，Ca）、磷（phosphorus，P）、羟脯氨酸（hydroxyproline，Hyp）、碱性磷酸酶（alkaline phosphatase，ALP）和抗酒石酸酸性磷酸酶（tartrate-resistant acid phosphatase，TRAP），同时采用蛋白质印迹法检测去势大鼠股骨TGF-β、p-Smad-3蛋白的表达以及p-JNK、p-ERK、p-P38、p-NF-κB P65蛋白的表达。结果 与正常组比较，模型组大鼠股骨Ca、P、Hyp、ALP水平显著下降（P<0.01），TRAP水平显著升高（P<0.01），骨小梁结构排列紊乱、形态变薄且有断裂迹象，TGF-β、p-Smad-3蛋白表达水平下调以及p-JNK、p-ERK、p-P38与p-NF-κB P65蛋白表达水平上调（P<0.01）。与模型组比较，龟鹿二仙胶显著增加大鼠股骨Ca、P、Hyp、ALP水平（P<0.01），降低股骨中TRAP水平（P<0.01），改善股骨病理改变，同时龟鹿二仙胶显著诱导TGF-β、p-Smad-3蛋白表达并显著降低p-JNK、p-ERK、p-P38与p-NF-κB P65蛋白表达（P<0.01）。结论 龟鹿二仙胶能显著改善去势大鼠的骨质疏松，其机制与激活TGF-β/Smad信号通路和抑制MAPK/NF-κB信号通路有关。

Abstract:

OBJECTIVE To evaluate the protective effect of Guilu Erxian Jiao on osteoporosis in ovariectomized rats and to explore its mechanism. METHODS A total of 120 rats were randomly divided into normal group, model group, low-dose, medium-dose and high-dose of Guilu Erxian Jiao group and alfacalcidol group. There were 6 groups in total, and all of them were emasculated and modeled except the normal group. Guilu Erxian Jiao was administrated with low, medium, high dose(0.5, 1.5, 2.5 g·kg^-1·d^-1), alfacalcidol was administrated with 0.1 μg·kg^-1·d^-1 dose, the normal group and the model group were given the same amount of normal saline respectively. The pathological changes of femur in rats were detected by HE staining, and the calcium(Ca), phosphorus(P), hydroxyproline(Hyp), alkaline phosphatase(ALP) and tartrate-resistant acid phosphatase(TRAP) of femur were detected by kits. At the same time, Western blotting was used to detect the expression of TGF-β, p-Smad-3 protein and p-JNK, p-ERK, p-P38, p-NF-κB P65 protein in femur of ovariectomized rats. RESULTS Compared with the normal group, the levels of Ca, P, Hyp and ALP in femur of rats in the model group were significantly decreased(P<0.01), while the level of TRAP was significantly increased(P<0.01). The trabecular structure of rats in the model group was disordered, the morphology was thinned and showed signs of fracture. The protein expression levels of TGF-β and p-Smad-3 in femur of the model group were down-regulated, while the protein expression levels of p-JNK, p-ERK, p-P38 and p-NF-κBP65 in femur were up-regulated(P<0.01). Compared with the model group, Guilu Erxian Jiao significantly increased the levels of Ca, P, Hyp and ALP in femur of rats(P<0.01), decreased the level of TRAP in femur of rats(P<0.01), and improved the pathological changes of femur. Meanwhile, Guilu Erxian Jiao significantly induced the expression of TGF-β and p-Smad-3 protein, and significantly decreased the expression of p-JNK, p-ERK, p-P38 and p-NF-κBP65 protein(P<0.01). CONCLUSION Guilu Erxian Jiao can significantly improve osteoporosis in ovariectomized rats, and the mechanism is related to the activation of TGF-β/Smad signaling pathway and the inhibition of MAPK/NF-κB signaling pathway.