去卵巢和雌二醇预防性治疗影响大鼠甲状旁腺中PTH基因及其关键调控因子的mRNA表达
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摘要
目的探讨雌激素和甲状旁腺素(parathyroid hormone,PTH)在绝经后骨质疏松症(postmenopausal osteoporosis,PMOP)发生、发展中的作用及其调控关系,为女性绝经后骨质疏松症的防治提供理论基础。通过构建去卵巢大鼠骨质疏松模型和雌二醇预防性治疗,分析大鼠甲状旁腺中PTH基因及其关键调控因子的mRNA表达。方法对去卵巢组(OVX)、雌二醇预防性治疗组(OVX/E_2)和假手术组(Sham)中大鼠的股骨与胫骨进行石蜡组织切片、苏木精-伊红染色(hematoxylin-eosin staining,H&E)和组织形态学观察。利用酶联免疫法和微孔板比色法测定大鼠血清雌激素和血清钙的含量变化。采用qPCR技术检测不同处理组间大鼠甲状旁腺中PTH、ER-α、CaSR、FGF-23、FGFR1、Klotho和Egr1基因的mRNA表达量变化。结果摘除卵巢后大鼠PTH的mRNA表达量显著降低,而预防性注射一定剂量的β-雌二醇能够显著提高PTH的mRNA表达量。研究发现摘除卵巢能显著提高甲状旁腺中ER-α的表达量,而雌二醇预防性治疗可以显著降低该基因的表达,提示甲状旁腺组织中的ER-α可能介导雌激素对PTH的表达调控。不同处理组之间,大鼠甲状旁腺中CaSR、FGF-23、FGFR1、Klotho和Egr1(调控PTH表达的潜在通路中的关键基因)的mRNA表达量的存在显著性差异,表明去卵巢和雌二醇预防性治疗影响了大鼠甲状旁腺中PTH关键调控因子的mRNA表达。结论去卵巢和一定剂量的β-雌二醇预防性给药会影响大鼠PTH基因的表达,大鼠甲状旁腺中的ER-α、Ca~(2+)/CaSR调控系统以及FGF-23-Klotho受体调控系统均可能参与PTH基因的表达调控。
Objective To explore the role and regulatory relationship of estrogen and parathyroid hormone(PTH) in the occurrence and development of postmenopausal osteoporosis(PMOP), the ovariectomized rat model of osteoporosis was used to analyze the effect of ovariectomy and prophylactic administration of estrogen on the regulation of PTH gene expression in PMOP rats. This will provide a theoretical basis for the prevention and treatment of postmenopausal osteoporosis in women. Methods The histological morphology of femur and tibia of rats in sham operation(Sham) group, ovariectomized(OVX) group and β-estradiol-treated OVX(OVX/E_2) group was observed in H& E-stained paraffin sections. The changes of serum estrogen and calcium in rats were determined by enzyme-linked immunosorbent assay(ELISA) and microporous plate colorimetry. The mRNA expression of PTH, ER-α, CaSR, FGF-23, FGFR1, Klotho and Egr1 genes in rat parathyroid glands were detected by real-time quantitative PCR(qPCR). Results The expression of PTH mRNA in ovariectomized rats was significantly decreased, while the expression of PTH mRNA was significantly increased by prophylactic administration of a certain dose of β-estradiol. It was found that the expression of ER-α was significantly up-regulated in rat parathyroid glands after ovariectomization, but significantly down-regulated after prophylactic administration of β-estradiol. It indicated that ER-α in parathyroid tissues may mediate the regulation of PTH expression by estrogen. The expression of CaSR, FGF-23, FGFR1, Klotho and Egr1, the key genes in the potential regulatory pathway of PTH expression, were significantly up-regulated or down-regulated in the parathyroid glands of rats in different treatment groups. These result suggest that ovariectomy and prophylactic estrogen therapy may affect the mRNA expression of key regulatory factors of PTH expression in the rat parathyroid glands. Conclusion Ovariectomy and prophylactic estrogen therapy can affect the expression of PTH gene. Estrogen receptor ER-α, Ca~(2+)/CaSR, and FGF-23-Klotho receptor regulatory system in rat parathyroid glands may participate in regulating PTH gene expression.
Objective To explore the role and regulatory relationship of estrogen and parathyroid hormone(PTH) in the occurrence and development of postmenopausal osteoporosis(PMOP), the ovariectomized rat model of osteoporosis was used to analyze the effect of ovariectomy and prophylactic administration of estrogen on the regulation of PTH gene expression in PMOP rats. This will provide a theoretical basis for the prevention and treatment of postmenopausal osteoporosis in women. Methods The histological morphology of femur and tibia of rats in sham operation(Sham) group, ovariectomized(OVX) group and β-estradiol-treated OVX(OVX/E_2) group was observed in H& E-stained paraffin sections. The changes of serum estrogen and calcium in rats were determined by enzyme-linked immunosorbent assay(ELISA) and microporous plate colorimetry. The mRNA expression of PTH, ER-α, CaSR, FGF-23, FGFR1, Klotho and Egr1 genes in rat parathyroid glands were detected by real-time quantitative PCR(qPCR). Results The expression of PTH mRNA in ovariectomized rats was significantly decreased, while the expression of PTH mRNA was significantly increased by prophylactic administration of a certain dose of β-estradiol. It was found that the expression of ER-α was significantly up-regulated in rat parathyroid glands after ovariectomization, but significantly down-regulated after prophylactic administration of β-estradiol. It indicated that ER-α in parathyroid tissues may mediate the regulation of PTH expression by estrogen. The expression of CaSR, FGF-23, FGFR1, Klotho and Egr1, the key genes in the potential regulatory pathway of PTH expression, were significantly up-regulated or down-regulated in the parathyroid glands of rats in different treatment groups. These result suggest that ovariectomy and prophylactic estrogen therapy may affect the mRNA expression of key regulatory factors of PTH expression in the rat parathyroid glands. Conclusion Ovariectomy and prophylactic estrogen therapy can affect the expression of PTH gene. Estrogen receptor ER-α, Ca~(2+)/CaSR, and FGF-23-Klotho receptor regulatory system in rat parathyroid glands may participate in regulating PTH gene expression.
引文
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[12] Reyes-Ibarra AP,Garcia-Regalado A,Ramirez-Rangel I,et al.Calcium-sensing receptor endocytosis links extracellular calcium signaling to parathyroid hormone-related peptide secretion via a Rab11a-dependent and AMSH-sensitive mechanism[J].Mol Endocrinol,2007,21(6):1394-1407.
[13] Fan Y,Liu W,Bi R,et al.Interrelated role of Klotho and calcium-sensing receptor in parathyroid hormone synthesis and parathyroid hyperplasia[J].Proc Natl Acad Sci USA,2018,115(16):E3749-E3758.
[14] 沈耿杨,任辉,江晓兵,等.去卵巢大鼠不同时期骨量、骨转换指标、雌激素水平的变化规律及相关性[J].中国组织工程研究,2015,19(2):170-176.
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[16] Lim SK,Won YJ,Lee HC,et al.A PCR analysis of ERα and ERβ mRNA abundance in rats and the effect of ovariectomy[J].J Bone Miner Res,1999,14(7):1189-1196.
[17] Stock JL,Coderre JA,Burke EM,et al.Identification of estrogen receptor mRNA and the estrogen modulation of parathyroid hormone-stimulated cyclic AMP accumulation in opossum kidney cells[J].J Cell Physiol,1992,150(3):517-525.
[18] Kanatani M,Sugimoto T,Takahashi Y,et al.Estrogen via the estrogen receptor blocks cAMP-mediated parathyroid hormone (PTH)-stimulated osteoclast formation[J].J Bone Miner Res,1998,13(5):854-862.
[19] Haglund F,Ma R,Huss M,et al.Evidence of a functional estrogen receptor in parathyroid adenomas[J].J Clin Endocrinol Metab,2012,97(12):4631-4639.
[20] 谢肇,李起鸿,孟萍,等.去卵巢大鼠骨质疏松模型的特点[J].中国临床康复,2006,10(28):79-81.
[21] 朱彤彤,黄连弟,李俊玮,等.淫羊藿苷对去卵巢大鼠骨质疏松症的保护作用及其机制[J].吉林大学学报(医学版),2016,42(5):915-919.
[22] Canalejo R,Canalejo A,Martinez-Moreno JM,et al.FGF23 fails to inhibit uremic parathyroid glands[J].J Am Soc Nephrol,2010,21(7):1125-1135.
[23] Galitzer H,Ben-Dov IZ,Silver J,et al.Parathyroid cell resistance to fibroblast growth factor 23 in secondary hyperparathyroidism of chronic kidney disease[J].Kidney Int,2010,77(3):211-218.
[2] 张婷婷,李游山,白瑜,等.雌激素预防性给药对去卵巢大鼠骨和脏器的影响[J].中国骨质疏松杂志,2017,23(12):1541-1545,1555.
[3] Ascenzi MG,Liao VP,Lee BM,et al.Parathyroid hormone treatment improves the cortical bone microstructure by improving the distribution of type I collagen in postmenopausal women with osteoporosis[J].J Bone Miner Res,2012,27(3):702-712.
[4] Pepe J,Cipriani C,Cantatore FP,et al.The effect of parathyroid hormone (1-84) treatment on serum bone morphogenetic protein 4 and vascular endothelial growth factor in postmenopausal women with established osteoporosis[J].J Endocrinol Invest,2017,40(6):663-667.
[5] Song J,Jin Z,Chang F,et al.Single and combined use of human parathyroid hormone (PTH) (1-34) on areal bone mineral density (aBMD) in postmenopausal women with osteoporosis:evidence based on 9 RCTs[J].Med Sci Monit,2014(20):2624-2632.
[6] Goltzman D,Hendy GN.The calcium-sensing receptor in bone--mechanistic and therapeutic insights[J].Nat Rev Endocrinol,2015,11(5):298-307.
[7] Kato S,Kim MS,Yamaoka K,et al.Mechanisms of transcriptional repression by 1,25(OH)2 vitamin D[J].Curr Opin Nephrol Hypertens,2007,16(4):297-304.
[8] Kim MS,Fujiki R,Murayama A,et al.1α,25(OH)2D3-induced transrepression by vitamin D receptor through E-box-type elements in the human parathyroid hormone gene promoter[J].Mol Endocrinol,2007,21(2):334-342.
[9] Ben-Dov IZ,Galitzer H,Lavi-Moshayoff V,et al.The parathyroid is a target organ for FGF23 in rats[J].J Clin Invest,2007,117(12):4003-4008.
[10] Livak KJ,Schmittgen TD.Analysis of relative gene expression data using real-time quantitative PCR and the 2 Method[J].Methods,2001,25(4):402-408.
[11] Brown EM,Gamba G,Riccardi D,et al.Cloning and characterization of an extracellular Ca2+-sensing receptor from bovine parathyroid[J].Nature,1993,366(6455):575-580.
[12] Reyes-Ibarra AP,Garcia-Regalado A,Ramirez-Rangel I,et al.Calcium-sensing receptor endocytosis links extracellular calcium signaling to parathyroid hormone-related peptide secretion via a Rab11a-dependent and AMSH-sensitive mechanism[J].Mol Endocrinol,2007,21(6):1394-1407.
[13] Fan Y,Liu W,Bi R,et al.Interrelated role of Klotho and calcium-sensing receptor in parathyroid hormone synthesis and parathyroid hyperplasia[J].Proc Natl Acad Sci USA,2018,115(16):E3749-E3758.
[14] 沈耿杨,任辉,江晓兵,等.去卵巢大鼠不同时期骨量、骨转换指标、雌激素水平的变化规律及相关性[J].中国组织工程研究,2015,19(2):170-176.
[15] 赵秀丽,贾三庆.雌激素与冠心病[M].北京:中国环境科学出版社,1997.
[16] Lim SK,Won YJ,Lee HC,et al.A PCR analysis of ERα and ERβ mRNA abundance in rats and the effect of ovariectomy[J].J Bone Miner Res,1999,14(7):1189-1196.
[17] Stock JL,Coderre JA,Burke EM,et al.Identification of estrogen receptor mRNA and the estrogen modulation of parathyroid hormone-stimulated cyclic AMP accumulation in opossum kidney cells[J].J Cell Physiol,1992,150(3):517-525.
[18] Kanatani M,Sugimoto T,Takahashi Y,et al.Estrogen via the estrogen receptor blocks cAMP-mediated parathyroid hormone (PTH)-stimulated osteoclast formation[J].J Bone Miner Res,1998,13(5):854-862.
[19] Haglund F,Ma R,Huss M,et al.Evidence of a functional estrogen receptor in parathyroid adenomas[J].J Clin Endocrinol Metab,2012,97(12):4631-4639.
[20] 谢肇,李起鸿,孟萍,等.去卵巢大鼠骨质疏松模型的特点[J].中国临床康复,2006,10(28):79-81.
[21] 朱彤彤,黄连弟,李俊玮,等.淫羊藿苷对去卵巢大鼠骨质疏松症的保护作用及其机制[J].吉林大学学报(医学版),2016,42(5):915-919.
[22] Canalejo R,Canalejo A,Martinez-Moreno JM,et al.FGF23 fails to inhibit uremic parathyroid glands[J].J Am Soc Nephrol,2010,21(7):1125-1135.
[23] Galitzer H,Ben-Dov IZ,Silver J,et al.Parathyroid cell resistance to fibroblast growth factor 23 in secondary hyperparathyroidism of chronic kidney disease[J].Kidney Int,2010,77(3):211-218.
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