左归丸调节β_2AR介导的RANKL/OPG信号通路改善去卵巢大鼠骨量和显微结构水平
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摘要
目的:探讨左归丸治疗绝经后骨质疏松症的分子机制之一是通过对β_2-肾上腺素能受体(β_2AR)介导的核转录因子-κB受体激活因子配体(RANKL)/骨保护素(OPG)信号通路的调节。方法:通过去卵巢法建立骨质疏松模型,将40只雌性SPF级SD大鼠随机分为模型组、假手术组、雌二醇(50μg·kg~(-1)·d~(-1))组、左归丸低、高剂量(2. 3,4. 6 g·kg~(-1))组。采用酶联免疫吸附实验检测血清骨转换标志物;显微CT对右侧股骨远端骨密度水平和骨小梁微结构进行测量和分析;实时荧光定量聚合酶链式反应(Real-time PCR)和蛋白免疫印迹法(Western blot)检测胫骨和下丘脑组织中β_2AR,OPG,RANK mRNA和蛋白表达水平。结果:与模型组比较,左归丸可显著降低大鼠血清中Ⅰ型胶原交联羧基端肽(β-CTX)水平(P <0. 01),增加骨特异性碱性磷酸酶(BALP)水平(P <0. 01),减少卵巢摘除诱导引起的大鼠骨量丢失,改善股骨远端骨小梁显微结构水平。同时,左归丸高、低剂量组均能显著上调胫骨组织中OPG mRNA和蛋白表达(P <0. 01),下调下丘脑组织β_2AR mRNA和蛋白表达水平(P <0. 01),同时也能显著下调胫骨组织中RANKL mRNA和蛋白表达水平(P <0. 01)。结论:左归丸改善围绝经期综合症,增加骨量和改善骨小梁显微结构水平的分子机制可能与对β_2AR介导的RANKL/OPG信号通路的调节有关。
Objective: To investigate the mechanism of Zuoguiwan in treating ovariectomy-induced osteoporosis rats by receptor activator of nuclear factor kappa-B ligand( RANKL)/osteoprotegerin( OPG)signaling pathway mediated by β_2-adrenergic receptor( β_2 AR). Method: Forty Sprague-Dawley female rats were randomly divided into Sham-operated group( Sham) and four ovariectomized( OVX) subgroups. Rats in Sham and OVX groups were treated with 17β-estradiol( 50 μg·kg~(-1)·d~(-1)),and low and high-dose ZGW( 2. 3,4. 6 g·kg~(-1) lyophilized powder) for 3 months,respectively. Enzyme-linked immunosorbent assay( ELISA) was used to measure the serum markers of bone turnover. Micro-CT was used to evaluate and measure trabecular bone microarchitecture and bone mineral density( BMD) of the right distal femur. Western blot analysis and Real-time PCR were used to measure mRNA and protein expressions of β_2 AR,OPG and RANKL. Result: After 12 weeks of treatment with Zuoguiwan,the level of serum β-cross-linked c-telopeptide of type Ι collagen( β-CTX)( P < 0. 01)was lower,while the level of serum bone-specific alkaline phosphatase( BALP) was higher( P < 0. 01) than those in the OVX group. Moreover,it could prevent the OVX-induced bone loss,and alleviate the trabecular bone microarchitecture of distal femur. Furthermore,Zuoguiwan could up-regulate the mRNA and protein expressions of OPG in tibia of the Zuoguiwan groups( P < 0. 01),reduce the mRNA and protein expressions of β_2 AR in the hypothalamus( P < 0. 01),and down-regulated the mRNA and protein expressions of RANKL( P < 0. 05) in the tibia,compared with those in the OVX group. Conclusion: The mechanism of Zuoguiwan in alleviating BMD and trabecular bone microarchitecture in ovariectomy-induced osteoporosis rats might be related to the regulation of RANKL/OPG Pathway mediated by β_2 AR.
Objective: To investigate the mechanism of Zuoguiwan in treating ovariectomy-induced osteoporosis rats by receptor activator of nuclear factor kappa-B ligand( RANKL)/osteoprotegerin( OPG)signaling pathway mediated by β_2-adrenergic receptor( β_2 AR). Method: Forty Sprague-Dawley female rats were randomly divided into Sham-operated group( Sham) and four ovariectomized( OVX) subgroups. Rats in Sham and OVX groups were treated with 17β-estradiol( 50 μg·kg~(-1)·d~(-1)),and low and high-dose ZGW( 2. 3,4. 6 g·kg~(-1) lyophilized powder) for 3 months,respectively. Enzyme-linked immunosorbent assay( ELISA) was used to measure the serum markers of bone turnover. Micro-CT was used to evaluate and measure trabecular bone microarchitecture and bone mineral density( BMD) of the right distal femur. Western blot analysis and Real-time PCR were used to measure mRNA and protein expressions of β_2 AR,OPG and RANKL. Result: After 12 weeks of treatment with Zuoguiwan,the level of serum β-cross-linked c-telopeptide of type Ι collagen( β-CTX)( P < 0. 01)was lower,while the level of serum bone-specific alkaline phosphatase( BALP) was higher( P < 0. 01) than those in the OVX group. Moreover,it could prevent the OVX-induced bone loss,and alleviate the trabecular bone microarchitecture of distal femur. Furthermore,Zuoguiwan could up-regulate the mRNA and protein expressions of OPG in tibia of the Zuoguiwan groups( P < 0. 01),reduce the mRNA and protein expressions of β_2 AR in the hypothalamus( P < 0. 01),and down-regulated the mRNA and protein expressions of RANKL( P < 0. 05) in the tibia,compared with those in the OVX group. Conclusion: The mechanism of Zuoguiwan in alleviating BMD and trabecular bone microarchitecture in ovariectomy-induced osteoporosis rats might be related to the regulation of RANKL/OPG Pathway mediated by β_2 AR.
引文
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[7]童伟伟.基于补肾益癸理论探讨左归丸防治PMOP的疗效机制[D].南京:南京中医药大学,2018.
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[11] LIU F,TAN F,TONG W,et al. Effect of Zuoguiwan on osteoporosis in ovariectomized rats through RANKL/OPG pathway mediated byβ2AR[J]. Biomed Pharmacoth,2018,103:1052-1060.
[12]吴佳莹,李岳泽,刘红,等.“肾主骨”机理研究—左归丸对Hepcidin、Fpn1及OPG/RANKL mRNA表达的影响[J].中国中医基础医学杂志,2017,23(11):1548-1551.
[13] JIANG H, WU Y, Valverde P, et al. Central adiponectin induces trabecular bone mass partly through epigenetic downregulation of cannabinoid receptor CB1[J]. J Cell Physiol,2019,234(5):7062-7069.
[14] Clément-Demange L,Mulcrone P L,Tabarestani T Q,et al.β2ARs stimulation in osteoblasts promotes breast cancer cell adhesion to bone marrow endothelial cells in an IL-1βand selectin-dependent manner[J]. J Bone Oncol,2018,13:1-10.
[15] Elefteriou F, Ahn J D, Takeda S, et al. Leptin regulation of bone resorption by the sympathetic nervous system and CART[J]. Nature,2005,434(7032):514-520.
[16] Noh H,YU M R,Kim H J,et al. Beta 2-adrenergic receptor agonists are novel regulators of macrophage activation in diabetic renal and cardiovascular complications[J]. Kidney Int,2017,92(1):101-113.
[17] MA Y,Nyman J S,TAO H,et al.β2-Adrenergic receptor signaling in osteoblasts contributes to the catabolic effect of glucocorticoids on bone[J].Endocrinology,2011,152(4):1412-1422.
[18] Mohammadpour H,O'Neil R,QIU J,et al. Blockade of hostβ2-adrenergic receptor enhances graft-versus-tumor effect through modulating APCs[J]. J Immunol,2018,200(7):2479-2488.
[19] Lee H,Choue R,Lim H. Effect of soy isoflavones supplement on climacteric symptoms,bone biomarkers,and quality of life in Korean postmenopausal women:a randomized clinical trial[J]. Nutr Res Pract,2017,11(3):223-231.
[20]梁文娜,李冠慧,林雪娟,等.二至丸调节Sirt1/Runx2信号通路抑制绝经后骨质疏松症肾阴虚证骨代谢失衡的机制探讨[J].中华中医药杂志,2018,33(7):2767-2769.
[21] Johnston B D,Ward W E. The ovariectomized rat as a model for studying alveolar bone loss in postmenopausal women[J]. Biomed Res Int,2015,2015:635023.
[22] Parfitt A M,Drezner M K,Glorieux F H,et al. Bone histomorphometry:standardization of nomenclature,symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee[J]. J Bone Miner Res,2009,2(6):595-610.
[23] Fu P,Gibson C J,Mendes W B,et al. Anxiety,depressive symptoms,and cardiac autonomic function in perimenopausal and postmenopausal women with hot flashes:a brief report[J]. Menopause,2018,25(12):1470-1475.
[24] Kruse N T,Hughes W E,Hanada S,et al. Evidence of a greater functional sympatholysis in habitually aerobic trained postmenopausal women[J]. J Appl Physiol,2018,124(3):583-591.
[25] Judex S,Pongkitwitoon S. Differential efficacy of 2vibrating orthodontic devices to alter the cellular response in osteoblasts,fibroblasts,and osteoclasts[J].Dose Response,2018,16(3):1559325818792112.
[26] Akbari Moghaddam Kakhki R,LU Z,Thanabalan A,et al. Eimeria challenge adversely affected long bone attributes linked to increased resorption in 14-day-old broiler chickens[J]. Poult Sci, 2019,98(4):1615-1621.
[27] ZHANG K,LI B,CHEN Q,et al. Functional calcium binding peptides from pacific cod(gadus macrocephalus)bone:calcium bioavailability enhancing activity and anti-osteoporosis effects in the ovariectomyinduced osteoporosis rat model[J]. Nutrients,2018,10(9):1325.
[28] Szulc P,Naylor K,Hoyle N R,et al. Use of CTX-I and PINP as bone turnover markers:National Bone Health Alliance recommendations to standardize sample handling and patient preparation to reduce pre-analytical variability[J]. Osteoporos Int, 2017, 28(9):2541-2556.
[29] Kanis J A,Cooper C,Rizzoli R,et al. Review of the guideline of the American College of Physicians on the treatment of osteoporosis[J]. Osteoporos Int,2018,29(7):1505-1510.
[30] Hauk L. Treatment of low BMD and osteoporosis to prevent fractures:updated guideline from the ACP[J].Am Fam Physician,2018,97(5):352-353.
[31] Hanson N A, Bagi C M. Alternative approach to assessment of bone quality using micro-computed tomography[J]. Bone,2004,35(1):326-333.
[32] Costa C, Eixarch H, Martínez-Sáez E, et al.Expression of bone morphogenetic proteins in multiple sclerosis lesions[J]. Am J Pathol,2018,189(3):665-676.
[33] Mulcrone P L,Campbell J P,Clément-Demange L,et al. Skeletal colonization by breast cancer cells is stimulated by an osteoblast andβ2AR-dependent neoangiogenic switch[J]. J Bone Miner Res,2017,32(7):1442-1454.
[34]尚德阳,邓洋洋,孙鑫,等.补肾中药对肾虚骨质疏松症大鼠骨、肾、下丘脑组织中Smurf1/Smurf2的mRNA表达影响[J].中华中医药杂志,2015,30(10):3629-3633.
[2] Hernlund E, Svedbom A, Ivergrd M, et al.Osteoporosis in the European Union:medical management,epidemiology and economic burden. A report prepared in collaboration with the International Osteoporosis Foundation(IOF)and the European Federation of Pharmaceutical Industry Associations(EFPIA)[J]. Arch Osteoporos,2013,8:136.
[3] Reid I R. Efficacy,effectiveness and side effects of medications used to prevent fractures[J]. J Intern Med,2015,277(6):690-706.
[4] Voss P J,Steybe D,Poxleitner P,et al. Osteonecrosis of the jaw in patients transitioning from bisphosphonates to denosumab treatment for osteoporosis[J]. Odontology,2018,6(4):469-480.
[5]张景岳.景岳全书[M].李继明,王大淳整理.北京:中国中医药出版社,2011:1283-1284.
[6]王如然,鞠大宏,黄胜男,等.左归丸治疗2型糖尿病合并骨质疏松肾阴虚证30例临床观察[J].中国中医基础医学杂志,2014,20(2):259-261.
[7]童伟伟.基于补肾益癸理论探讨左归丸防治PMOP的疗效机制[D].南京:南京中医药大学,2018.
[8]余新莲.左归丸合下瘀血汤治疗原发性骨质疏松症(肾虚血瘀型)临床疗效观察[J].中医临床研究,2016,8(33):10-11.
[9]魏志敏.左归丸加减联合钙剂治疗肝肾阴虚型骨质疏松症近期临床疗效分析[D].成都:成都中医药大学,2015.
[10]谭峰,樊巧玲,卞玉群,等.左、右归丸对去卵巢骨质疏松症大鼠模型骨密度及骨代谢的影响[J].中国实验方剂学杂志,2015,21(9):137-140.
[11] LIU F,TAN F,TONG W,et al. Effect of Zuoguiwan on osteoporosis in ovariectomized rats through RANKL/OPG pathway mediated byβ2AR[J]. Biomed Pharmacoth,2018,103:1052-1060.
[12]吴佳莹,李岳泽,刘红,等.“肾主骨”机理研究—左归丸对Hepcidin、Fpn1及OPG/RANKL mRNA表达的影响[J].中国中医基础医学杂志,2017,23(11):1548-1551.
[13] JIANG H, WU Y, Valverde P, et al. Central adiponectin induces trabecular bone mass partly through epigenetic downregulation of cannabinoid receptor CB1[J]. J Cell Physiol,2019,234(5):7062-7069.
[14] Clément-Demange L,Mulcrone P L,Tabarestani T Q,et al.β2ARs stimulation in osteoblasts promotes breast cancer cell adhesion to bone marrow endothelial cells in an IL-1βand selectin-dependent manner[J]. J Bone Oncol,2018,13:1-10.
[15] Elefteriou F, Ahn J D, Takeda S, et al. Leptin regulation of bone resorption by the sympathetic nervous system and CART[J]. Nature,2005,434(7032):514-520.
[16] Noh H,YU M R,Kim H J,et al. Beta 2-adrenergic receptor agonists are novel regulators of macrophage activation in diabetic renal and cardiovascular complications[J]. Kidney Int,2017,92(1):101-113.
[17] MA Y,Nyman J S,TAO H,et al.β2-Adrenergic receptor signaling in osteoblasts contributes to the catabolic effect of glucocorticoids on bone[J].Endocrinology,2011,152(4):1412-1422.
[18] Mohammadpour H,O'Neil R,QIU J,et al. Blockade of hostβ2-adrenergic receptor enhances graft-versus-tumor effect through modulating APCs[J]. J Immunol,2018,200(7):2479-2488.
[19] Lee H,Choue R,Lim H. Effect of soy isoflavones supplement on climacteric symptoms,bone biomarkers,and quality of life in Korean postmenopausal women:a randomized clinical trial[J]. Nutr Res Pract,2017,11(3):223-231.
[20]梁文娜,李冠慧,林雪娟,等.二至丸调节Sirt1/Runx2信号通路抑制绝经后骨质疏松症肾阴虚证骨代谢失衡的机制探讨[J].中华中医药杂志,2018,33(7):2767-2769.
[21] Johnston B D,Ward W E. The ovariectomized rat as a model for studying alveolar bone loss in postmenopausal women[J]. Biomed Res Int,2015,2015:635023.
[22] Parfitt A M,Drezner M K,Glorieux F H,et al. Bone histomorphometry:standardization of nomenclature,symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee[J]. J Bone Miner Res,2009,2(6):595-610.
[23] Fu P,Gibson C J,Mendes W B,et al. Anxiety,depressive symptoms,and cardiac autonomic function in perimenopausal and postmenopausal women with hot flashes:a brief report[J]. Menopause,2018,25(12):1470-1475.
[24] Kruse N T,Hughes W E,Hanada S,et al. Evidence of a greater functional sympatholysis in habitually aerobic trained postmenopausal women[J]. J Appl Physiol,2018,124(3):583-591.
[25] Judex S,Pongkitwitoon S. Differential efficacy of 2vibrating orthodontic devices to alter the cellular response in osteoblasts,fibroblasts,and osteoclasts[J].Dose Response,2018,16(3):1559325818792112.
[26] Akbari Moghaddam Kakhki R,LU Z,Thanabalan A,et al. Eimeria challenge adversely affected long bone attributes linked to increased resorption in 14-day-old broiler chickens[J]. Poult Sci, 2019,98(4):1615-1621.
[27] ZHANG K,LI B,CHEN Q,et al. Functional calcium binding peptides from pacific cod(gadus macrocephalus)bone:calcium bioavailability enhancing activity and anti-osteoporosis effects in the ovariectomyinduced osteoporosis rat model[J]. Nutrients,2018,10(9):1325.
[28] Szulc P,Naylor K,Hoyle N R,et al. Use of CTX-I and PINP as bone turnover markers:National Bone Health Alliance recommendations to standardize sample handling and patient preparation to reduce pre-analytical variability[J]. Osteoporos Int, 2017, 28(9):2541-2556.
[29] Kanis J A,Cooper C,Rizzoli R,et al. Review of the guideline of the American College of Physicians on the treatment of osteoporosis[J]. Osteoporos Int,2018,29(7):1505-1510.
[30] Hauk L. Treatment of low BMD and osteoporosis to prevent fractures:updated guideline from the ACP[J].Am Fam Physician,2018,97(5):352-353.
[31] Hanson N A, Bagi C M. Alternative approach to assessment of bone quality using micro-computed tomography[J]. Bone,2004,35(1):326-333.
[32] Costa C, Eixarch H, Martínez-Sáez E, et al.Expression of bone morphogenetic proteins in multiple sclerosis lesions[J]. Am J Pathol,2018,189(3):665-676.
[33] Mulcrone P L,Campbell J P,Clément-Demange L,et al. Skeletal colonization by breast cancer cells is stimulated by an osteoblast andβ2AR-dependent neoangiogenic switch[J]. J Bone Miner Res,2017,32(7):1442-1454.
[34]尚德阳,邓洋洋,孙鑫,等.补肾中药对肾虚骨质疏松症大鼠骨、肾、下丘脑组织中Smurf1/Smurf2的mRNA表达影响[J].中华中医药杂志,2015,30(10):3629-3633.