模拟海拔5000米低氧环境下大鼠甲状腺轴与CRH家族的相关性研究
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摘要
目的研究SD大鼠HPT轴激素及蛋白在低氧环境下的病理生理变化机制及与促肾上腺皮质激素释放激素(Corticotropin releasing hormone,CRH)家族相关蛋白及激素的相互作用。方法用低压低氧舱模拟海拔5 000 m高原低氧环境复制SD大鼠动物模型,采用ELISA法检测大鼠血清中HPT轴激素水平,包括游离三碘甲腺原氨酸(Free triido-thyronine,FT3)、游离四碘甲腺原氨酸(Free tetraiodo-thyronine,FT4)、总三碘甲腺原氨酸(Total triido-thyronine,TT3)、总四碘甲腺原氨酸(Total tetraiodo-thyronine,TT4)、反三碘甲腺原氨酸(3,5,3’-L-triiodo-thyronine,r T3)、促甲状腺激素(Thyrotropin stimulating hormone,TSH)、促甲状腺激素释放激素(Thyrotropin releasing hormone,TRH),以及CRH家族激素CRH、尿皮素2(Urocortin II,Ucn2)、Ucn3、皮质酮(Corticosterone,CORT)在血清中的水平,通过Western blot检测低氧诱导因子-1α(Hypoxia-inducible factor 1α,HIF-1α),HPT轴的TSHR、TRHR1及CRH家族受体CRHR1、CRHR2的表达。采用免疫荧光双染检测CRHR1、CRHR2与TSHR在甲状腺滤泡细胞的表达。通过Pearson相关分析探讨CRH家族CRH、Ucn2、Ucn3、CORT和HPT轴激素FT3、FT4、TT3、TT4、TSH、TRH、r T3相关性。结果低氧组大鼠TRH、TT3、TT4、FT3、FT4和TSH血清水平均下降(P<0.05),r T3、CRH、Ucn2、Ucn3和CORT血清浓度上升(P<0.05)。相关性统计表明低氧各组大鼠血清CRH与TRH激素水平负相关(r=-0.80),Ucn2与FT3、TT3分别呈负相关(r=-0.796,-0.798)。免疫荧光双标显示CRHR1与TSHR、CRHR2与TSHR在甲状腺滤泡细胞存在共表达。结论结果表明模拟5000米海拔低氧环境使大鼠HPT轴功能受到抑制,可能与CRH家族基因激活有关,这对进一步研究HPT轴与CRH家族相互作用机制提供一定的线索。
Objective To explore the changes of HPT axis hormones and proteins, and counterpart of CRH family, then investigate the association between their changes under the hypoxia conditions. Methods We explored the effect of hypoxia by establishing animal model by hypobaric chamber with simulated altitude 5 000 m. HPT axis hormones FT3, FT4, TT3, TT4, r T3, TSH,TRH and CRH family-related hormones CRH, Ucn2, Ucn3, CORT were tected in serum by ELISA. The expression of HIF-1α, HPT axis key factors TSHR, TRHR1 and CRH family-related genes CRHR1, CRHR2 were detected by Western blot. The colocalization of CRHR1, CRHR2 and TSHR in thyroid follicular cells were tested by Immunofluorescence. The correlation of CRH, Ucn2, Ucn3,CORT and FT3, FT4, TT3, TT4, TSH, TRH, r T3 were statistially analyzed by Pearson. Results Exposure to hypoxia equivalent to an altitude of 5000 m, HPT axis hormones TRH, TT3, TT4, FT3, FT4 and TSH were decreased(P<0.05) and r T3, CRH, Ucn2, Ucn3 and CORT were increase(P<0.05) under hypoxia. The levels of CRH, Ucn2 were negatively correlated with TRH, FT3 and TT3 respectively(r=-0.80,-0.796,-0.798). Immunofluorescence staining showed coexpression of CRHR1 and TSHR, CRHR2 and TSHR in thyroid follicular cells. Conclusion The results showed that the HPT axis function was inhibited in rats under simulated 5000 meters altitude, which may relate to the activation of CRH family genes. The study may provide some clues for the further study of the interaction mechanism between the HPT axis and the CRH family.
Objective To explore the changes of HPT axis hormones and proteins, and counterpart of CRH family, then investigate the association between their changes under the hypoxia conditions. Methods We explored the effect of hypoxia by establishing animal model by hypobaric chamber with simulated altitude 5 000 m. HPT axis hormones FT3, FT4, TT3, TT4, r T3, TSH,TRH and CRH family-related hormones CRH, Ucn2, Ucn3, CORT were tected in serum by ELISA. The expression of HIF-1α, HPT axis key factors TSHR, TRHR1 and CRH family-related genes CRHR1, CRHR2 were detected by Western blot. The colocalization of CRHR1, CRHR2 and TSHR in thyroid follicular cells were tested by Immunofluorescence. The correlation of CRH, Ucn2, Ucn3,CORT and FT3, FT4, TT3, TT4, TSH, TRH, r T3 were statistially analyzed by Pearson. Results Exposure to hypoxia equivalent to an altitude of 5000 m, HPT axis hormones TRH, TT3, TT4, FT3, FT4 and TSH were decreased(P<0.05) and r T3, CRH, Ucn2, Ucn3 and CORT were increase(P<0.05) under hypoxia. The levels of CRH, Ucn2 were negatively correlated with TRH, FT3 and TT3 respectively(r=-0.80,-0.796,-0.798). Immunofluorescence staining showed coexpression of CRHR1 and TSHR, CRHR2 and TSHR in thyroid follicular cells. Conclusion The results showed that the HPT axis function was inhibited in rats under simulated 5000 meters altitude, which may relate to the activation of CRH family genes. The study may provide some clues for the further study of the interaction mechanism between the HPT axis and the CRH family.
引文
1 Rajoria S,Hanly E,Nicolini A,et al.Interlinking of hypoxia and estrogen in thyroid cancer progression[J].Curr Med Chem,2014,21(11):1351-1360.
2 Timiras PS.Hypoxia and the CNS:maturation and adaptation at high altitude[J].Int J Biometeorol,1977,21(2):147-156.
3吕静,孙小康,赵长明,等.急进高原暴露下皮质醇变化的研究进展[J].西南军医,2014,(6):676-678.
4 Shi ZX,Levy A,Lightman SL.Thyroid hormone-mediated regulation of corticotropin-releasing hormone messenger ribonucleic acid in the rat[J].Endocrinology,1994,134(3):1577-1580.
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7 Sawhney RC,Malhotra AS.Thyroid function during intermittent exposure to hypobaric hypoxia[J].Int J Biometeorol,1990,34(3):161-173.
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9彭华,李素芝,王洪斌.缺氧对健康成人甲状腺功能的影响[J].西南国防医药,2006,(6):643-644.
10 Richalet JP,Letournel M,Souberbielle JC.Effects of highaltitude hypoxia on the hormonal response to hypothalamic factors[J].Am J Physiol Regul Integr Comp Physiol,2010,299(6):R1685-1692.
11 Gradwell E.Histological changes in the thyroid gland in rats on acclimatisation to simulated high altitude[J].JPathol,1978,125(1):33-37.
12 Reinhardt W,Mocker V,Jockenh?vel F,et al.Influence of coronary artery bypass surgery on thyroid hormone parameters[J].Horm Res,1997,47(1):1-8.
13 Koob GF.Corticotropin-releasing factor,norepinephrine,and stress[J].Biol Psychiatry,1999,46(9):1167-1180.
14 Barnholt KE,Hoffman AR,Rock PB,et al.Endocrine responses to acute and chronic high-altitude exposure(4,300 meters):modulating effects of caloric restriction[J].Am J Physiol Endocrinol Metab,2006,290(6):E1078-1088.
15 Boelen A,Maas MA,Lowik CW,et al.Induced illness in interleukin-6(IL-6)knock-out mice:a causal role of IL-6in the development of the low 3,5,3′-triiodothyronine syndrome[J].Endocrinology,1996,137(12):5250-5264.
16 Richalet JP,Letournel M,Souberbielle JC.Effects of highaltitude hypoxia on the hormonal response to hypothalamic factors[J].Am J Physiol Regul Integr Comp Physiol,2010,299(6):R1685-1692.
17周珺,贾正平等.模拟高原低氧应激环境下大鼠内分泌代谢的变化研究[J].西北国防医学杂志同,2014.20(4):304-308.
2 Timiras PS.Hypoxia and the CNS:maturation and adaptation at high altitude[J].Int J Biometeorol,1977,21(2):147-156.
3吕静,孙小康,赵长明,等.急进高原暴露下皮质醇变化的研究进展[J].西南军医,2014,(6):676-678.
4 Shi ZX,Levy A,Lightman SL.Thyroid hormone-mediated regulation of corticotropin-releasing hormone messenger ribonucleic acid in the rat[J].Endocrinology,1994,134(3):1577-1580.
5 Helmreich DL,Parfitt DB,Lu XY,et al.Relation between the hypothalamic-pituitary-thyroid(HPT)axis and the hypothalamic-pituitary-adrenal(HPA)axis during repeated stress[J].Neuroendocrinology,2005,81(3):183-192.
6 Squillacioti C,De Luca A,AlìS,et al.Presence and distribution of urocortin and corticotrophin-releasing hormone receptors in the bovine thyroid gland[J].Anat Histol Embryol,2014,43(6):429-434.
7 Sawhney RC,Malhotra AS.Thyroid function during intermittent exposure to hypobaric hypoxia[J].Int J Biometeorol,1990,34(3):161-173.
8侯天德,杜继曾.模拟急性低氧大鼠血清T3,T4和下丘脑TRH含量变化[J].中国应用生理学杂志,2001,(1):9-10.
9彭华,李素芝,王洪斌.缺氧对健康成人甲状腺功能的影响[J].西南国防医药,2006,(6):643-644.
10 Richalet JP,Letournel M,Souberbielle JC.Effects of highaltitude hypoxia on the hormonal response to hypothalamic factors[J].Am J Physiol Regul Integr Comp Physiol,2010,299(6):R1685-1692.
11 Gradwell E.Histological changes in the thyroid gland in rats on acclimatisation to simulated high altitude[J].JPathol,1978,125(1):33-37.
12 Reinhardt W,Mocker V,Jockenh?vel F,et al.Influence of coronary artery bypass surgery on thyroid hormone parameters[J].Horm Res,1997,47(1):1-8.
13 Koob GF.Corticotropin-releasing factor,norepinephrine,and stress[J].Biol Psychiatry,1999,46(9):1167-1180.
14 Barnholt KE,Hoffman AR,Rock PB,et al.Endocrine responses to acute and chronic high-altitude exposure(4,300 meters):modulating effects of caloric restriction[J].Am J Physiol Endocrinol Metab,2006,290(6):E1078-1088.
15 Boelen A,Maas MA,Lowik CW,et al.Induced illness in interleukin-6(IL-6)knock-out mice:a causal role of IL-6in the development of the low 3,5,3′-triiodothyronine syndrome[J].Endocrinology,1996,137(12):5250-5264.
16 Richalet JP,Letournel M,Souberbielle JC.Effects of highaltitude hypoxia on the hormonal response to hypothalamic factors[J].Am J Physiol Regul Integr Comp Physiol,2010,299(6):R1685-1692.
17周珺,贾正平等.模拟高原低氧应激环境下大鼠内分泌代谢的变化研究[J].西北国防医学杂志同,2014.20(4):304-308.