基于肠源性5-HT介导的NKX2.2/Lmx1a信号轴揭示藤黄健骨胶囊对绝经后骨质疏松模型鼠的干预机制

Therapeutic Mechanism of Tenghuang Jiangu capsule on Rats with Postmenopausal Osteoporosis based on Enterogenous 5-HT Mediating NKX2.2/Lmx1a Signaling Axis

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

Key words:  postmenopausal osteoporosis  Tenghuang Jiangu capsule  5-HT  NKX2.2/Lmx1a signaling axis

摘要:

目的 通过观察藤黄健骨胶囊(Tenghuang Jiangu capsule,TJC)对绝经后骨质疏松模型鼠NKX2.2/ Lmx1a信号轴关键分子NKX2.2、Lmx1a和5-HT表达的影响,初步揭示其可能的治疗机制。SD雌性大鼠随机分为假手术组、OVX模型组、戊酸雌二醇组(0.09mg/kg)、藤黄健骨胶囊干预组(0.36、0.18、0.09g·kg?1)。OVX大鼠模型复制方法为摘卵巢法。建模8周后每组动物分别给予上述药物灌胃,连续干预时间为8周,干预结束后次日测定每组大鼠BMD和BMC,观察股骨病理形态改变；ELISA检测血清BSP和ATF4水平改变；免疫荧光检测每组大鼠十二指肠组NKX2.2、Lmx1a和5-HT表达；qPCR和Western-blotc测定每组大鼠十二指肠组织NKX2.2、Lmx1a、Tph1表达及股骨组织5-HT1BR和FOXO1表达；Western-blot检测每组大鼠股骨CREB表达。结果 与假手术组比较,OVX模型鼠股骨组织出现明显的病理损伤,BMD和BMC指标、血清BSP和ATF4含量和股骨组织CREB蛋白表达降低,而十二指肠组织NKX2.2、Lmx1a、Tph1基因表达则升高,NKX2.2、Lmx1a、5-HT的荧光强度及NKX2.2、Lmx1a、Tph1蛋白表达也升高,股骨组织5-HT1BR、FOXO1基因及蛋白表达水平也升高(P<0.01)。与OVX模型组比较,0.36g·kg?1TJC组股骨组织病理损伤明显减轻,0.18、0.36g·kg?1 TJC组大鼠血清BSP、ATF4含量及股骨组织CREB蛋白表达水平均增加和十二指肠组织NKX2.2、Tph1蛋白表达水平均降低,0.09、0.18、0.36g·kg?1TJC组大鼠BMD和BMC指标升高,而十二指肠NKX2.2、Lmx1a、Tph1基因表达和NKX2.2、Lmx1a、5-HT的荧光强度及Lmx1a蛋白表达水平均降低,股骨组织5-HT1BR、FOXO1基因及蛋白表达水平也均降低(P<0.05或P<0.01)。结论 藤黄健骨胶囊能显著上调绝经后骨质疏松模型鼠的促成骨分化因子BSP和ATF4的表达,这可能与抑制肠源性5-HT介导的NKX2.2/Lmx1a/5-HT信号轴有关。

Abstract:

ABSTRACT: OBJECTIVE To observing the effect of Tenghuang Jiangu capsule(TJC) on the expression of NKX2.2、Lmx1a and 5-HT of NKX2.2/Lmx1a signaling axis, which was explained its therapeutic mechanism. METHODS 60 female SD rats were randomly divided into sham operation group (10ml·kg?1 distilled water), ovariectomized model group(10ml·kg?1 distilled water), estradiol valerate group(0.09 mg·kg?1), intervention groups of TJC (the doses of 0.36, 0.18, 0.09 g·kg?1). Except for the sham operation group, rats in other groups were used to establish a rat model of postmenopausal osteoporosis by ovariectomized method. After 8 weeks of modeling, rats in each group were given above doses of drugs by oral gavage, with continuous intervention for 8 weeks. The following day after intervention, the levels of bone mineral density (BMD) and bone mineral content (BMC) were detected, the pathomorphological changes of femoral tissue was observed by Goldner's staining, the concentrations of bone sialoprotein(BSP) and transcription activator 4(ATF4) were checked by ELISA, immunofluorescence was used to detect the protein expressions of nkx2.2 homeodomain transcription factor(NKX2.2), Lim Homeobox transcription factor 1a(Lmx1a) and5-hydroxytryptamine(5-HT), the gene and protein expressions of NKX2.2、Lmx1a and tryptophan hydroxylase 1(Tph1) of duodenum tissue were analyzed by qPCR and Western-blot, the gene and protein expressions of 5-hydroxytryptamine receptor 1B(5-HT1BR), forkhead transcription factor-1(FOXO1) of femoral tissue were detected by qPCR and Western-blot, the protein expression of cAMP-response element binding protein(CREB) were detected by Western-blot. RESULTS Compared with the sham operation group, the femoral tissue of rats in the ovariectomized model group had obvious pathological changes, the levels of BMD, BMC, the concentrations of BSP and ATF4 of serum, and the protein expression level of CREB of femoral tissu were all significantly decreased, whereas the gene expression levels of NKX2.2, Lmx1a and Tph1 of duodenum tissue were significantly increased, the fluorescence intensity of NKX2.2, Lmx1a and 5-HT, the protein expression levels of NKX2.2, Lmx1a and Tph1were also significantly increased, and the gene and protein expression levels of 5-HT1BR and FOXO1 of femoral tissue were significantly increased (P<0.01). Compared with the ovariectomized model group, the pathological damage of femoral tissue was reduced in 0.36g·kg?1 TJC group, the concentrations of BSP and ATF4 of serum and the protein expression level of CREB of femoral tissue were all significantly increased in 0.18, 0.36g·kg?1 TJC groups, the protein expression levels of NKX2.2, Tph1 of duodenum tissue were significantly decreased in 0.18, 0.36g·kg?1 TJC groups, the levels of BMD and BMC of rats in 0.09, 0.18, 0.36g·kg?1 TJC groups were significantly increased, however the gene expression levels of NKX2.2, Lmx1a,Tph1, the fluorescence intensity of NKX2.2, Lmx1a and 5-HT, the protein expression level of Lmx1a of duodenum tissue were all significantly decreased in 0.09, 0.18, 0.36g·kg?1 TJC groups, the gene and protein expression levels of 5-HT1BR and FOXO1 of femoral tissue were also significantly decreased in 0.09, 0.18, 0.36g·kg?1 TJC groups(P<0.05 or P<0.01). CONCLUSION TJC could significantly upregulate the expression of bone differentiation factors BSP and ATF4 in postmenopausal osteoporosis rats, which maybe related to the inhibition of NKX2.2/Lmx1a/5-HT signaling axis by intestinal 5-HT mediated.