藤黄健骨胶囊通过NKX2.2/Lmx1b/5-HT通路调控绝经后骨质疏松症大鼠脑神经递质变化的实验研究

Experimental Study of Tenghuang Jiangu Capsule on Regulating the Changes of Cranial nerves Neurotransmitters in Postmenopausal Osteoporosis Rats through NKX2.2/Lmx1b/5-HT Pathway

关键词:  藤黄健骨胶囊  绝经后骨质疏松症  NKX2.2/Lmx1b/5-HT信号通路  

Key words:  Tenghuang Jiangu capsules  postmenopausal osteoporosis  NKX2.2/Lmx1b/5-HT signal pathway

摘要:

目的 探讨NKX2.2/Lmx1b/5-HT信号通路对绝经后骨质疏松症大鼠脑神经递质变化的影响,并揭示藤黄健骨胶囊可能的干预机制。方法 60只SPF级SD雌性大鼠,随机分为假手术组、模型组、戊酸雌二醇组(0.09g/kg)、藤黄健骨胶囊高、中、低剂量组(0.36、0.18、0.09g/kg),每组10只。除假手术组外,其余各组均采用摘除双侧卵巢法来建立绝经后骨质疏松症模型。模型制备成功后,假手术组和模型组给予等体积蒸馏水灌胃,戊酸雌二醇组和藤黄健骨胶囊各干预组分别给予相应剂量药物灌胃。干预8周后,股动脉采血并处死各组大鼠,收集脑和股骨组织备用。观察各组大鼠的一般状况和体重；HE染色观察各组大鼠股骨组织病理形态学改变；免疫荧光染色检测各组大鼠脑组织NKX2.2、Lmx1b和5-HT荧光表达强度；RT-PCR法、Western-Blot法检测各组大鼠脑组织NKX2.2、Lmx1b、Tph2、5-HT2CR和股骨组织β2AR基因和蛋白表达。结果 与假手术组比较,模型组和藤黄健骨胶囊各剂量组大鼠造模8w后体重均明显增加,模型组大鼠股骨组织骨小梁稀疏断裂、数目明显减少,有较多空隙、不能连接成网,梁髓比显著降低,骨髓脂肪相对面积(BMAT)显著升高(P<0.05或P<0.01)；股骨组织中β2AR基因表达则明显增加(P<0.01)；脑组织中NKX2.2、Lmx1b、5-HT平均荧光强度、NKX2.2、Lmx1b、Tph2和5-HT2CR基因和蛋白表达及蛋白表达灰度值均明显降低(P<0.01)。与模型组比较,藤黄健骨胶囊高剂量组大鼠干预8w后体重均明显降低,股骨组织骨小梁数量、排列及连接接近于假手术组,梁髓比显著升高(P<0.05或P<0.01)；藤黄健骨胶囊中、高剂量组大鼠股骨组织BMAT显著降低(P<0.01)；脑组织NKX2.2平均荧光强度和NKX2.2、Lmx1b、Tph2基因及Lmx1b、Tph2、5-HT2CR蛋白表达均明显升高(P<0.05或P<0.01)；藤黄健骨胶囊各剂量组大鼠脑组织Lmx1b和5-HT平均荧光强度、5-HT2CR基因表达、NKX2.2蛋白表达均明显升高,而股骨组织β2AR基因和蛋白及蛋白表达均明显降低(P<0.05或P<0.01)。结论 藤黄健骨胶囊通过激活PMOP大鼠脑组织中NKX2.2/Lmx1b/5-HT信号通路来改善其股骨病理形态损伤,从而发挥治疗PMOP的作用。

Abstract:

ABSTRACT: OBJECTIVE To explore the effect of NKX2.2/Lmx1b/5-HT signal pathway on the changes of cranial nerves transmitters in postmenopausal osteoporosis rats, and to reveal the possible intervention mechanism of Tenghuang Jiangu capsule. METHODS Sixty SPF SD female rats were randomly divided into a Sham surgery group, a model group, a estradiol valerate group (0.09 g/kg), and a high, medium, and low dose group of Tenghuang Jiangu capsule (0.36, 0.18, 0.09 g/kg), with 10 rats in each group. Except for the Sham surgery group, the other groups used bilateral ovariectomy to establish postmenopausal osteoporosis models. After the model was successfully prepared, the Sham surgery group and the model group were given equal volume of distilled water by gavage, and the sstradiol valerate group and the intervention groups of Tenghuang Jiangu capsule were given corresponding dose by gavage. After 8 weeks of intervention, the rats were killed by femoral artery, and brain and femur tissues were collected for reservation. The general condition and weight were observein each group. HE staining was used to observe the morphological change of rat femur. Immunofluorescence staining was used to detect the expression intensity of NKX2.2, Lmx1b, and 5-HT of rat brain. RT-PCR and Western Blot methods were used to detect the gene and protein expressions of NKX2.2, Lmx1b, Tph2, 5-HT2CR of rat brain and the gene and protein expressions of β2ARof rat femur. RESULTS Compared with the sham surgery group, the weight of rats in the model group and the Tenghuang Jiangu capsule groups increased significantly after modeling for 8 weeks. The number of bone trabeculae of rat femur was significantly reduced in the model group, with more gaps, and could not be connected into a network. The ratio of beam to pulp was significantly reduced, and the relative area of bone marrow fat (BMAT) was significantly increased (P<0.05 or P<0.01); The expression of β2AR gene significantly increased in femoral tissue (P<0.01). The average fluorescence intensity of NKX2.2, Lmx1b, 5-HT, and the gene and protein expressions of NKX2.2, Lmx1b, Tph2, and 5-HT2CR were all significantly reduced in brain tissue (P<0.01). Compared with the model group, the weight decreased significantly in the high dose group of Tenghuang Jiangu capsule after 8 weeks of intervention, the number, arrangement and connection of bone trabeculae in the femur tissue were close to those in the sham surgery group, and the ratio of beam to pulp was significantly increased (P<0.05 or P<0.01). BMAT of rat femur decreased significantly in middle and high dose groups of Tenghuang Jiangu capsule(P<0.01). The average fluorescence intensity of NKX2.2 in brain tissue, as well as the gene expressions of NKX2.2, Lmx1b, the protein expressions of Tph2, Lmx1b, Tph2, 5-HT2CR were all significantly increased (P<0.05 or P<0.01). The average fluorescence intensity of Lmx1b and 5-HT, the expression of 5-HT2CR gene, the expression of NKX2.2 protein in brain tissue of Tenghuang Jiangu capsule groups were significantly increased, however the gene and protein expressions of β2AR in the femoral tissue were significantly reduced (P<0.05 or P<0.01).CONCLUSION Tenghuang Jiangu capsule plays a therapeutic role by activating the NKX2.2/Lmx1b/ 5-HT signaling pathway in PMOP rats.